Polypeptide variants with altered effector function

ABSTRACT

The present invention concerns polypeptides comprising a variant Fc region. More particularly, the present invention concerns Fc region-containing polypeptides that have altered effector function as a consequence of one or more amino acid modifications in the Fc region thereof.

This is a divisional of U.S. application Ser. No. 12/590,801, filed Nov.12, 2009, which is a continuation of U.S. application Ser. No.11/520,121, filed Sep. 13, 2006, now abandoned, which is a continuationto continuation-in-part application Ser. No. 09/713,425 filed Nov. 15,2000, now U.S. Pat. No. 7,183,387 issued Feb. 27, 2007, which claimspriority to non-provisional application Ser. No. 09/483,588, filed Jan.14, 2000 (now U.S. Pat. No. 6,737,056 issued May 18, 2004), which claimspriority under 35 USC §119 to provisional application No. 60/116,023filed Jan. 15, 1999, the entire disclosures of which are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention concerns polypeptides comprising a variant Fcregion. More particularly, the present invention concerns Fcregion-containing polypeptides that have altered effector function as aconsequence of one or more amino acid modifications in the Fc regionthereof.

2. Description of Related Art

Antibodies are proteins which exhibit binding specificity to a specificantigen. Native antibodies are usually heterotetrameric glycoproteins ofabout 150,000 daltons, composed of two identical light (L) chains andtwo identical heavy (H) chains. Each light chain is linked to a heavychain by one covalent disulfide bond, while the number of disulfidelinkages varies between the heavy chains of different immunoglobulinisotypes. Each heavy and light chain also has regularly spacedintrachain disulfide bridges. Each heavy chain has at one end a variabledomain (V_(H)) followed by a number of constant domains. Each lightchain has a variable domain at one end (V_(L)) and a constant domain atits other end; the constant domain of the light chain is aligned withthe first constant domain of the heavy chain, and the light chainvariable domain is aligned with the variable domain of the heavy chain.Particular amino acid residues are believed to form an interface betweenthe light and heavy chain variable domains.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areresponsible for the binding specificity of each particular antibody forits particular antigen. However, the variability is not evenlydistributed through the variable domains of antibodies. It isconcentrated in three segments called complementarity determiningregions (CDRs) both in the light chain and the heavy chain variabledomains. The more highly conserved portions of the variable domains arecalled the framework regions (FRs). The variable domains of native heavyand light chains each comprise four FRs, largely adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FRs and, with the CDRsfrom the other chain, contribute to the formation of the antigen bindingsite of antibodies (see Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)).

The constant domains are not involved directly in binding an antibody toan antigen, but exhibit various effector functions. Depending on theamino acid sequence of the constant region of their heavy chains,antibodies or immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG andIgM, and several of these may be further divided into subclasses(isotypes), e.g. IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2. The heavychain constant regions that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. Of thevarious human immunoglobulin classes, only human IgG1, IgG2, IgG3 andIgM are known to activate complement; and human IgG1 and IgG3 mediateADCC more effectively than IgG2 and IgG4.

A schematic representation of the native IgG1 structure is shown in FIG.1, where the various portions of the native antibody molecule areindicated. Papain digestion of antibodies produces two identical antigenbinding fragments, called Fab fragments, each with a single antigenbinding site, and a residual “Fc” fragment, whose name reflects itsability to crystallize readily. The crystal structure of the human IgGFc region has been determined (Deisenhofer, Biochemistry 20:2361-2370(1981)). In human IgG molecules, the Fc region is generated by papaincleavage N-terminal to Cys 226. The Fc region is central to the effectorfunctions of antibodies.

The effector functions mediated by the antibody Fc region can be dividedinto two categories: (1) effector functions that operate after thebinding of antibody to an antigen (these functions involve theparticipation of the complement cascade or Fc receptor (FcR)-bearingcells); and (2) effector functions that operate independently of antigenbinding (these functions confer persistence in the circulation and theability to be transferred across cellular barriers by transcytosis).Ward and Ghetie, Therapeutic Immunology 2:77-94 (1995).

While binding of an antibody to the requisite antigen has a neutralizingeffect that might prevent the binding of a foreign antigen to itsendogenous target (e.g. receptor or ligand), binding alone may notremove the foreign antigen. To be efficient in removing and/ordestructing foreign antigens, an antibody should be endowed with bothhigh affinity binding to its antigen, and efficient effector functions.

Fc Receptor (FcR) Binding

The interaction of antibodies and antibody-antigen complexes with cellsof the immune system effects a variety of responses, includingantibody-dependent cell-mediated cytotoxicity (ADCC) and complementdependent cytotoxicity (CDC) (reviewed in Daëron, Annu. Rev. Immunol.15:203-234 (1997); Ward and Ghetie, Therapeutic Immunol. 2:77-94 (1995);as well as Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991)).

Several antibody effector functions are mediated by Fc receptors (FcRs),which bind the Fc region of an antibody. FcRs are defined by theirspecificity for immunoglobulin isotypes; Fc receptors for IgG antibodiesare referred to as FcγR, for IgE as FcεR, for IgA as FcαR and so on.Three subclasses of FcγR have been identified: FcγRI (CD64), FcγRII(CD32) and FcγRIII (CD16). Because each FcγR subclass is encoded by twoor three genes, and alternative RNA spicing leads to multipletranscripts, a broad diversity in FcγR isoforms exists. The three genesencoding the FcγRI subclass (FcγRIA, FcγRIB and FcγRIC) are clustered inregion 1q21.1 of the long arm of chromosome 1; the genes encoding FcγRIIisoforms (FcγRIIA, FcγRIIB and FcγRIIC) and the two genes encodingFcγRIII (FcγRIIIA and FcγRIIIB) are all clustered in region 1q22. Thesedifferent FcR subtypes are expressed on different cell types (reviewedin Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991)). Forexample, in humans, FcγRIIIB is found only on neutrophils, whereasFcγRIIIA is found on macrophages, monocytes, natural killer (NK) cells,and a subpopulation of T-cells. Notably, FcγRIIIA is the only FcRpresent on NK cells, one of the cell types implicated in ADCC.

FcγRI, FcγRII and FcγRIII are immunoglobulin superfamily (IgSF)receptors; FcγRI has three IgSF domains in its extracellular domain,while FcγRII and FcγRIII have only two IgSF domains in theirextracellular domains.

Another type of Fc receptor is the neonatal Fc receptor (FcRn). FcRn isstructurally similar to major histocompatibility complex (MHC) andconsists of an α-chain noncovalently bound to β2-microglobulin.

The binding site on human and murine antibodies for FcγR have beenpreviously mapped to the so-called “lower hinge region” consisting ofresidues 233-239 (EU index numbering as in Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Woof et al. Molec.Immunol. 23:319-330 (1986); Duncan et al. Nature 332:563 (1988);Canfield and Morrison, J. Exp. Med. 173:1483-1491 (1991); Chappel etal., Proc. Natl. Acad. Sci USA 88:9036-9040 (1991). Of residues 233-239,P238 and S239 have been cited as possibly being involved in binding, butthese two residues have never been evaluated by substitution ordeletion.

Other previously cited areas possibly involved in binding to FcγR are:G316-K338 (human IgG) for human FcγRI (by sequence comparison only; nosubstitution mutants were evaluated) (Woof et al. Molec. Immunol.23:319-330 (1986)); K274-R301 (human IgG1) for human FcγRIII (based onpeptides) (Sarmay et al. Molec. Immunol. 21:43-51 (1984)); Y407-R416(human IgG) for human FcγRIII (based on peptides) (Gergely et al.Biochem. Soc. Trans. 12:739-743 (1984)); as well as N297 and E318(murine IgG2b) for murine FcγRII (Lund et al., Molec. Immunol., 29:53-59(1992)).

Pro331 in IgG3 was changed to Ser, and the affinity of this variant totarget cells analyzed. The affinity was found to be six-fold lower thanthat of unmutated IgG3, indicating the involvement of Pro331 in FcγRIbinding. Morrison et al., Immunologist, 2:119-124 (1994); and Canfieldand Morrison, J. Exp. Med. 173:1483-91 (1991).

C1q Binding

C1q and two serine proteases, C1r and C1s, form the complex C1, thefirst component of the complement dependent cytotoxicity (CDC) pathway.C1q is a hexavalent molecule with a molecular weight of approximately460,000 and a structure likened to a bouquet of tulips in which sixcollagenous “stalks” are connected to six globular head regions. Burtonand Woof, Advances in Immunol. 51:1-84 (1992). To activate thecomplement cascade, it is necessary for C1q to bind to at least twomolecules of IgG1, IgG2, or IgG3 (the consensus is that IgG4 does notactivate complement), but only one molecule of IgM, attached to theantigenic target. Ward and Ghetie, Therapeutic Immunology 2:77-94 (1995)at page 80.

Based upon the results of chemical modifications and crystallographicstudies, Burton et al. (Nature, 288:338-344 (1980)) proposed that thebinding site for the complement subcomponent C1q on IgG involves thelast two (C-terminal) β-strands of the CH2 domain. Burton latersuggested (Molec. Immunol., 22(3):161-206 (1985)) that the regioncomprising amino acid residues 318 to 337 might be involved incomplement fixation.

Duncan and Winter (Nature 332:738-40 (1988)), using site directedmutagenesis, reported that Glu318, Lys320 and Lys322 form the bindingsite to C1q. The data of Duncan and Winter were generated by testing thebinding of a mouse IgG2b isotype to guinea pig C1q. The role of Glu318,Lys320 and Lys322 residues in the binding of C1q was confirmed by theability of a short synthetic peptide containing these residues toinhibit complement mediated lysis. Similar results are disclosed in U.S.Pat. No. 5,648,260 issued on Jul. 15, 1997, and U.S. Pat. No. 5,624,821issued on Apr. 29, 1997.

The residue Pro331 has been implicated in C1q binding by analysis of theability of human IgG subclasses to carry out complement mediated celllysis. Mutation of Ser331 to Pro331 in IgG4 conferred the ability toactivate complement. (Tao et al., J. Exp. Med., 178:661-667 (1993);Brekke et al., Eur. J. Immunol., 24:2542-47 (1994)).

From the comparison of the data of the Winter group, and the Tao et al.and Brekke et al. papers, Ward and Ghetie concluded in their reviewarticle that there are at least two different regions involved in thebinding of C1q: one on the β-strand of the CH2 domain bearing theGlu318, Lys320 and Lys322 residues, and the other on a turn located inclose proximity to the same β-strand, and containing a key amino acidresidue at position 331.

Other reports suggested that human IgG1 residues Leu235, and Gly237,located in the lower hinge region, play a critical role in complementfixation and activation. Xu et al., Immunol. 150:152A (Abstract) (1993).WO94/29351 published Dec. 22, 1994 reports that amino acid residuesnecessary for C1q and FcR binding of human IgG1 are located in theN-terminal region of the CH2 domain, i.e. residues 231 to 238.

It has further been proposed that the ability of IgG to bind C1q andactivate the complement cascade also depends on the presence, absence,or modification of the carbohydrate moiety positioned between the twoCH2 domains (which is normally anchored at Asn297). Ward and Ghetie,Therapeutic Immunology 2:77-94 (1995) at page 81.

SUMMARY OF THE INVENTION

The present invention provides a variant of a parent polypeptidecomprising an Fc region, which variant mediates antibody-dependentcell-mediated cytotoxicity (ADCC) in the presence of human effectorcells more effectively, or binds an Fc gamma receptor (FcγR) with betteraffinity, than the parent polypeptide and comprises at least one aminoacid modification in the Fc region. The polypeptide variant may, forexample, comprise an antibody or an immunoadhesin. The Fc region of theparent polypeptide preferably comprises a human Fc region; e.g., a humanIgG1, IgG2, IgG3 or IgG4 Fc region. The polypeptide variant preferablycomprises an amino acid modification (e.g. a substitution) at any one ormore of amino acid positions 256, 290, 298, 312, 326, 330, 333, 334,360, 378 or 430 of the Fc region, wherein the numbering of the residuesin the Fc region is that of the EU index as in Kabat.

In addition, the invention provides a polypeptide comprising a variantFc region with altered Fc gamma receptor (FcγR) binding affinity, whichpolypeptide comprises an amino acid modification at any one or more ofamino acid positions 238, 239, 248, 249, 252, 254, 255, 256, 258, 265,267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292,293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322,324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373,376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or439 of the Fc region, wherein the numbering of the residues in the Fcregion is that of the EU index as in Kabat. The variant Fc regionpreferably comprises a variant human IgG Fc region, e.g., a varianthuman IgG1, IgG2, IgG3 or IgG4 Fc region. In this respect, it is notedthat, in the work in the above-cited art where the parent polypeptidehad a non-human murine Fc region, different residues from thoseidentified herein were thought to impact FcR binding. For example, inthe murine IgG2b/murine FcγRII system, IgG E318 was found to beimportant for binding (Lund et al. Molec. Immunol. 27(1):53-59 (1992)),whereas E318A had no effect in the human IgG/human FcγRII system (Table6 below).

In one embodiment, the polypeptide variant with altered FcγR bindingactivity displays reduced binding to an FcγR and comprises an amino acidmodification at any one or more of amino acid positions 238, 239, 248,249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293, 294, 295,296, 298, 301, 303, 322, 324, 327, 329, 333, 335, 338, 340, 373, 376,382, 388, 389, 414, 416, 419, 434, 435, 437, 438 or 439 of the Fcregion, wherein the numbering of the residues in the Fc region is thatof the EU index as in Kabat.

For example, the polypeptide variant may display reduced binding to anFcγRI and comprise an amino acid modification at any one or more ofamino acid positions 238, 265, 269, 270, 327 or 329 of the Fc region,wherein the numbering of the residues in the Fc region is that of the EUindex as in Kabat.

The polypeptide variant may display reduced binding to an FcγRII andcomprise an amino acid modification at any one or more of amino acidpositions 238, 265, 269, 270, 292, 294, 295, 298, 303, 324, 327, 329,333, 335, 338, 373, 376, 414, 416, 419, 435, 438 or 439 of the Fcregion, wherein the numbering of the residues in the Fc region is thatof the EU index as in Kabat.

The polypeptide variant of interest may display reduced binding to anFcγRIII and comprise an amino acid modification at one or more of aminoacid positions 238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272,278, 289, 293, 294, 295, 296, 301, 303, 322, 327, 329, 338, 340, 373,376, 382, 388, 389, 416, 434, 435 or 437 of the Fc region, wherein thenumbering of the residues in the Fc region is that of the EU index as inKabat.

In another embodiment, the polypeptide variant with altered FcγR bindingaffinity displays improved binding to the FcγR and comprises an aminoacid modification at any one or more of amino acid positions 255, 256,258, 267, 268, 272, 276, 280, 283, 285, 286, 290, 298, 301, 305, 307,309, 312, 315, 320, 322, 326, 330, 331, 333, 334, 337, 340, 360, 378,398 or 430 of the Fc region, wherein the numbering of the residues inthe Fc region is that of the EU index as in Kabat.

For example, the polypeptide variant may display increased binding to anFcγRIII and, optionally, may further display decreased binding to anFcγRII. An exemplary such variant comprises amino acid modification(s)at position(s) 298 and/or 333 of the Fc region, wherein the numbering ofthe residues in the Fc region is that of the EU index as in Kabat.

The polypeptide variant may display increased binding to an FcγRII andcomprise an amino acid modification at any one or more of amino acidpositions 255, 256, 258, 267, 268, 272, 276, 280, 283, 285, 286, 290,301, 305, 307, 309, 312, 315, 320, 322, 326, 330, 331, 337, 340, 378,398 or 430 of the Fc region, wherein the numbering of the residues inthe Fc region is that of the EU index as in Kabat. Such polypeptidevariants with increased binding to an FcγRII may optionally furtherdisplay decreased binding to an FcγRIII and may, for example, comprisean amino acid modification at any one or more of amino acid positions268, 272, 298, 301, 322 or 340 of the Fc region, wherein the numberingof the residues in the Fc region is that of the EU index as in Kabat.

The invention further provides a polypeptide comprising a variant Fcregion with altered neonatal Fc receptor (FcRn) binding affinity, whichpolypeptide comprises an amino acid modification at any one or more ofamino acid positions 238, 252, 253, 254, 255, 256, 265, 272, 286, 288,303, 305, 307, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380,382, 386, 388, 400, 413, 415, 424, 433, 434, 435, 436, 439 or 447 of theFc region, wherein the numbering of the residues in the Fc region isthat of the EU index as in Kabat. Such polypeptide variants with reducedbinding to an FcRn may comprise an amino acid modification at any one ormore of amino acid positions 252, 253, 254, 255, 288, 309, 386, 388,400, 415, 433, 435, 436, 439 or 447 (and preferably one or more of aminoacid positions 253, 254, 435 or 436) of the Fc region, wherein thenumbering of the residues in the Fc region is that of the EU index as inKabat. The above mentioned polypeptide variants may, alternatively,display increased binding to FcRn and comprise an amino acidmodification at any one or more of amino acid positions 238, 256, 265,272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378,380, 382, 413, 424 or 434 (and preferably one or more of amino acidpositions 238, 256, 307, 311, 312, 380, 382 or 434) of the Fc region,wherein the numbering of the residues in the Fc region is that of the EUindex as in Kabat.

The polypeptide variant may be in one of the following classes ofvariants as shown in Table 6 herein (with increased, reduced orunchanged binding to a receptor being calculated as described withrespect to that table):

1. A polypeptide variant which displays reduced binding to FcγRI,FcγRIIA, FcγRIIB and FcγRIIIA comprising an amino acid modification atany one or more of amino acid positions 238, 265, 327 or 329 of the Fcregion, wherein the numbering of the residues in the Fc region is thatof the EU index as in Kabat.

2. A polypeptide variant with reduced binding to FcγRII and FcγRIIIAcomprising an amino acid modification at any one or more of amino acidpositions 270, 295 or 327 of the Fc region, wherein the numbering of theresidues in the Fc region is that of the EU index as in Kabat.

3. A polypeptide variant which displays increased binding to FcγRII andFcγRIIIA comprising an amino acid modification at any one or more ofamino acid positions 256 or 290 of the Fc region, wherein the numberingof the residues in the Fc region is that of the EU index as in Kabat.

4. A polypeptide variant which displays increased binding to FcγRII butunchanged binding to FcγRIIIA and comprises an amino acid modificationat any one or more of amino acid positions 255, 258, 267, 272, 276, 280,285, 286, 307, 309, 315, 326, 331, 337, 378 or 430 of the Fc region,wherein the numbering of the residues in the Fc region is that of the EUindex as in Kabat.

5. A polypeptide variant which displays increased binding to FcγRII andreduced binding to FcγRIIIA comprising an amino acid modification at anyone or more of amino acid positions 268, 301 or 322 of the Fc region,wherein the numbering of the residues in the Fc region is that of the EUindex as in Kabat.

6. A polypeptide variant which displays reduced binding to FcγRII butunchanged FcγRIIIA binding and comprises an amino acid modification atany one or more of amino acid positions 292 or 414 of the Fc region,wherein the numbering of the residues in the Fc region is that of the EUindex as in Kabat.

7. A polypeptide variant which displays reduced binding to FcγRII andimproved binding to FcγRIIIA comprising an amino acid modification atamino acid position 298 of the Fc region, wherein the numbering of theresidues in the Fc region is that of the EU index as in Kabat.

8. A polypeptide variant which displays reduced binding to FcγRIIIA butunchanged FcRII binding comprising an amino acid modification at any oneor more of amino acid positions 239, 269, 293, 296, 303, 327, 338 or 376of the Fc region, wherein the numbering of the residues in the Fc regionis that of the EU index as in Kabat.

9. A polypeptide variant which displays improved binding to FcγRIIIA butunchanged FcRII binding comprising an amino acid modification at any oneor more of amino acid positions 333 or 334 of the Fc region, wherein thenumbering of the residues in the Fc region is that of the EU index as inKabat.

10. A polypeptide variant which displays altered binding to FcRn butunchanged FcγR binding and comprises an amino acid modification at anyone or more of amino acid positions 253, 254, 288, 305, 311, 312, 317,360, 362, 380, 382, 415, 424, 433, 434, 435 or 436 of the Fc region,wherein the numbering of the residues in the Fc region is that of the EUindex as in Kabat.

The polypeptide variant herein with improved ADCC activity is preferablyselected from:

1. A polypeptide variant which comprises an amino acid modification atany one or more of amino acid positions 298, 333 or 334 of the Fcregion, wherein the numbering of the residues in the Fc region is thatof the EU index as in Kabat.

2. A polypeptide variant which comprises amino acid modifications at twoor more of amino acid positions 298, 333, 334 or 339 of the FC region,wherein the numbering of the residues in the Fc region is that of the EUindex as in Kabat.

3. A polypeptide variant which comprises an amino acid modification atamino acid position 298 of the Fc region, wherein the numbering of theresidues in the Fc region is that of the EU index as in Kabat.

4. A polypeptide variant which comprises amino acid modifications atamino acid positions 298 and 334 of the Fc region, wherein the numberingof the residues in the Fc region is that of the EU index as in Kabat.

5. A polypeptide variant which comprises amino acid modifications atthree or more of amino acid positions 298, 333, 334 or 339 of the Fcregion, wherein the numbering of the residues in the Fc region is thatof the EU index as in Kabat.

6. A polypeptide variant which comprises amino acid modifications atamino acid positions 298, 333 and 334 of the Fc region, wherein thenumbering of the residues in the Fc region is that of the EU index as inKabat.

The invention provides a polypeptide variant selected from:

1. A polypeptide comprising a variant Fc region which comprises aminoacid modifications at amino acid positions 298, 333 and 334 of the Fcregion, wherein the numbering of the residues in the Fc region is thatof the EU index as in Kabat.

2. A polypeptide comprising a variant Fc region which comprises aminoacid modifications at amino acid positions 298 and 334 of the Fc region,wherein the numbering of the residues in the Fc region is that of the EUindex as in Kabat.

3. A polypeptide comprising a variant Fc region which comprises an aminoacid modification at amino acid position 298 of the Fc region, whereinthe numbering of the residues in the Fc region is that of the EU indexas in Kabat.

4. A polypeptide comprising a variant Fc region which comprises aminoacid modifications at two or all of amino acid positions 307, 380 and434 of the Fc region, wherein the numbering of the residues is that ofthe EU index as in Kabat.

In a further embodiment, the invention provides a polypeptide comprisinga variant Fc region with altered affinity for an FcγR allotype (e.g. anFcγRIIIA-Phe158, FcγRIIIA-Val158, FcγRIIA-R131 or FcγRIIA-H131allotype), which polypeptide comprises an amino acid modification at anyone or more of amino acid positions 265, 267, 268, 270, 290, 298, 305,307, 315, 317, 320, 331, 333 or 334 of the Fc region, wherein thenumbering of the residues in the Fc region is that of the EU index as inKabat. Preferably, the polypeptide displays increased binding toFcγRIIIA-Phe158 and hence has improved therapeutic effectiveness,particularly in patients who express FcγRIIIA-Phe158. Polypeptidevariants with increased binding to FcγRIIIA-Phe158 preferably comprisean amino acid modification at anyone or more of amino acid positions290, 298, 333 or 334 of the Fc region, wherein the numbering of theresidues in the Fc region is that of the EU index as in Kabat.

The invention also provides a composition comprising the polypeptidevariant and a physiologically or pharmaceutically acceptable carrier ordiluent. This composition for potential therapeutic use is sterile andmay be lyophilized.

Diagnostic and therapeutic uses for the polypeptide variants disclosedherein are contemplated. In one diagnostic application, the inventionprovides a method for determining the presence of an antigen of interestcomprising exposing a sample suspected of containing the antigen to thepolypeptide variant and determining binding of the polypeptide variantto the sample. In one therapeutic application, the invention provides amethod of treating a mammal suffering from or predisposed to a diseaseor disorder, comprising administering to the mammal a therapeuticallyeffective amount of a polypeptide variant as disclosed herein, or of acomposition comprising the polypeptide variant and a pharmaceuticallyacceptable carrier.

The invention further provides: isolated nucleic acid encoding thepolypeptide variant; a vector comprising the nucleic acid, optionally,operably linked to control sequences recognized by a host celltransformed with the vector; a host cell containing the vector; a methodfor producing the polypeptide variant comprising culturing this hostcell so that the nucleic acid is expressed and, optionally, recoveringthe polypeptide variant from the host cell culture (e.g. from the hostcell culture medium).

The invention further provides a method for making a variant Fc regionwith altered Fc receptor (FcR) binding affinity, or alteredantibody-dependent cell-mediated cytotoxicity (ADCC) activity,comprising:

(a) introducing one or more amino acid modifications into an Fc regionof a parent polypeptide in order to generate a variant Fc region;

(b) determining binding of the variant Fc region to an FcR, ordetermining ADCC activity of the variant Fc region.

Step (b) of the method may comprise determining binding of the variantFc region to one or more FcRs in vitro. Moreover, the method may resultin the identification of a variant Fc region with improved FcR bindingaffinity, or with improved ADCC activity, in step (b) thereof. Wherestep (b) comprises determining binding of the Fc region to an FcR, theFcR may, for example, be human Fc gamma receptor III (FcγRIII). Wherestep (b) comprises determining binding of the variant Fc region to atleast two different FcRs, the FcRs tested preferably include human Fcgamma receptor II (FcγRII) and human Fc gamma receptor III (FcγRIII).

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1 is a schematic representation of a native IgG. Disulfide bondsare represented by heavy lines between CH1 and CL domains and the twoCH2 domains. V is variable domain; C is constant domain; L stands forlight chain and H stands for heavy chain.

FIG. 2 shows C1q binding of wild type (wt) C2B8 antibody; C2B8 antibodywith a human IgG2 constant region (IgG2); and variants K322A, K320A andE318A.

FIG. 3 depicts C1q binding of variants P331A, P329A and K322A.

FIGS. 4A and 4B depict the amino acid sequences of E27 anti-IgE antibodylight chain (FIG. 4A; SEQ ID NO:1) and heavy chain (FIG. 4B; SEQ IDNO:2).

FIG. 5 is a schematic diagram of the “immune complex” prepared for usein the FcR assay described in Example 1. The hexamer comprising threeanti-IgE antibody molecules (the “Fc region-containing polypeptide”) andthree IgE molecules (the “first target molecule”) is shown. IgE has two“binding sites” for the anti-IgE antibody (E27) in the Fc regionthereof. Each IgE molecule in the complex is further able to bind twoVEGF molecules (“the second target polypeptide”). VEGF has two “bindingsites” for IgE.

FIG. 6 shows C1q binding results obtained for variants D270K and D270Vcompared to wild type C2B8.

FIG. 7 depicts complement dependent cytotoxicity (CDC) of variants D270Kand D270V, compared to wild type C2B8.

FIG. 8 shows C1q binding ELISA results for 293 cell-produced wild typeC2B8 antibody (293-Wt-C2B8), CHO-produced wild type C2B8 antibody(CHO-Wt-C2B8) and various variant antibodies.

FIG. 9 shows C1q binding ELISA results obtained for wild type (wt) C2B8and various variant antibodies as determined in Example 3.

FIG. 10 depicts the three-dimensional structure of a human IgG Fcregion, highlighting residues: Asp270, Lys326, Pro329, Pro331, Lys322and Glu333.

FIG. 11 shows C1q binding ELISA results obtained for wild type C2B8 andvarious variant antibodies as determined in Example 3.

FIG. 12 shows C1q binding ELISA results obtained for wild type C2B8 anddouble variants, K326M-E333S and K326A-E333A.

FIG. 13 shows CDC of wild type C2B8 and double variants, K326M-E333S andK326A-E333A.

FIG. 14 depicts C1q binding ELISA results obtained for C2B8 with a humanIgG4 (IgG4), wild type C2B8 (Wt-C2B8), C2B8 with a human IgG2 constantregion (IgG2), and variant antibodies as described in Example 3.

FIGS. 15A and 15B show binding patterns for parent antibody (E27) toFcγRIIB and FcγRIIIA. FIG. 15A shows the binding pattern for thehumanized anti-IgE E27 IgG1 as a monomer (open circles), hexamer (closedsquares), and immune complex consisting of multiple hexamers (closedtriangles) to a recombinant GST fusion protein of the human FcγRIIB(CD32) receptor α subunit. The hexameric complex (closed squares) wasformed by the mixture of equal molar concentrations of E27 (which bindsto the Fc region of human IgE) and a human myeloma IgE. The hexamer is astable 1.1 kD complex consisting of 3 IgG molecules (150 kD each) and 3IgE molecules (200 kD each). The immune complex (closed triangles) wasformed sequentially by first mixing equal molar concentrations of E27and recombinant anti-VEGF IgE (human IgE with Fab variable domains thatbind human VEGF) to form the hexamer. Hexamers were then linked to forman immune complex by the addition of 2× molar concentration of humanVEGF, a 44 kD homodimer which has two binding sites for the anti-VEGFIgE per mole of VEGF. FIG. 15B shows the binding pattern to arecombinant GST fusion protein of the human FcγRIIIA (CD16) receptor αsubunit.

FIG. 16A shows the binding of immune complexes using differentantigen-antibody pairs to recombinant GST fusion protein of the FcγRIIAreceptor α subunit. FIG. 16B shows the binding of the sameantigen-antibody pairs to the GST fusion protein of the FcγRIIIAreceptor α subunit. Closed circles represent binding of humanIgE:anti-IgE E27 IgG1; open circles represent binding of humanVEGF:humanized anti-VEGF IgG1.

FIG. 17 summarizes differences in binding selectivity of some alaninevariants between the different FcγRs. Binding of alanine variants atresidues in the CH2 domain of anti-IgE E27 IgG1 are shown to FcγRIIA,FcγRIIB, and FcγRIIIA. Type 1 abrogates binding to all three receptors:D278A (265 in EU numbering). Type 2 improves binding to FcγRIIA andFcγRIIB, while binding to FcγRIIIA is unaffected: S280A (267 in EUnumbering). Type 3 improves binding to FcγRIIA and FcγRIIB, but reducesbinding to FcγRIIIA: H281A (268 in EU numbering). Type 4 reduces bindingto FcγRIIA and FcγRIIB, while improving binding to FcγRIIIA: S317A (298in EU numbering). Type 5 Improves binding to FcγRIIIA, but does notaffect binding to FcγRIIA and FcγRIIB: E352A, K353A (333 and 334 in EUnumbering).

FIGS. 18A and 18B compare the FcγRIIIA protein/protein assay and CHOGPI-FcγRIIIA cell based assay, respectively. FIG. 18A illustratesbinding of selected alanine variants to FcγRIIIA-GST fusion protein.S317A (298 in EU numbering) and S317A/K353A (298 and 334 in EUnumbering) bind better than E27 wildtype, while D278A (265 in EUnumbering) almost completely abrogates binding. FIG. 18B illustratesthat a similar pattern of binding is found on CHO cells expressing arecombinant GPI-linked form of FcγRIIIA.

FIGS. 19A and 19B compare the FcγRIIB protein/protein assay and CHOGPI-FcγRIIB cell based assay, respectively. FIG. 19A illustrates bindingof selected alanine variants to FcγRIIB-GST fusion protein. H281A (268in EU numbering) binds better than E27 wildtype while S317A (298 in EUnumbering) shows reduced binding. FIG. 19B illustrates that a similarpattern of binding is found on CHO cells expressing a recombinantmembrane bound form of FcγRIIB.

FIG. 20 shows single alanine substitutions in the CH2 domain ofanti-HER2 IgG1 (HERCEPTIN®) that influence FcγRIIIA binding in both theprotein-protein and cell-based assays alter the ability to bind toFcγRIIIA on peripheral blood mononuclear cell (PBMC) effector cells.Recombinant humanized anti-HER2 (HERCEPTIN®), which binds toHER2-expressing SK-BR-3 breast tumor cells, was preincubated with⁵¹Cr-labeled SK-BR-3 cells for 30 minutes (opsonization) at 100 ng/ml(filled circles) and 1.25 ng/ml (filled squares). Keeping the SK-BR-3tumor target cell concentration constant, the ratio of effector cellswas increased from 0 to 100. The spontaneous cytotoxicity in the absenceof antibody (hatched squares) was 20% at an effector:target (E:T) ratioof 100:1. A single alanine mutation that did not affect FcγRIIIAbinding, variant G31=R309A (292 in EU numbering), did not effect ADCC(filled triangles). A single alanine mutation that only slightlyincreased binding to FcγRIIIA, variant G30=K307A (290 in EU numbering),also showed slightly improved ADCC (i.e., a 1.1 fold improvement in ADCCactivity, calculated as area under the curve) at 1.25 ng/ml at all E:Tratios (filled diamonds) compared to wildtype antibody at 1.25 ng/ml(filled square). A single alanine mutation that decreased binding toFcγRIIIA, variant G34=Q312A (295 in EU numbering), also showed decreasedADCC activity (filled inverted triangles).

FIG. 21 illustrates that a single alanine mutation which had the mostimproved binding to FcγRIIIA, variant G36=S317A (298 in EU numbering),in the protein-protein and cell-based assays also showed the mostimprovement in ADCC (filled triangles) among the variants compared towildtype (closed squares) at 1.25 ng/ml. G36 displayed a 1.7 foldimprovement in ADCC activity, calculated as area under the curve.Variants G17=E282A (269 in EU numbering) and G18=D283A (270 in EUnumbering) both showed reduced binding to FcγRIIIA as well as reducedefficacy in ADCC. The effector cells were PBMCs.

FIG. 22A depicts alignments of native sequence IgG Fc regions. Nativesequence human IgG Fc region sequences, humIgG1 (non-A and A allotypes)(SEQ ID NOs: 3 and 4, respectively), humIgG2 (SEQ ID NO:5), humIgG3 (SEQID NO:6) and humIgG4 (SEQ ID NO:7), are shown. The human IgG1 sequenceis the non-A allotype, and differences between this sequence and the Aallotype (at positions 356 and 358; EU numbering system) are shown belowthe human IgG1 sequence. Native sequence murine IgG Fc region sequences,murIgG1 (SEQ ID NO:8), murIgG2A (SEQ ID NO:9), murIgG2B (SEQ ID NO:10)and murIgG3 (SEQ ID NO:11), are also shown. FIG. 22B shows percentidentity among the Fc region sequences of FIG. 22A.

FIG. 23 depicts alignments of native sequence human IgG Fc regionsequences, humIgG1 (non-A and A allotypes; SEQ ID NOs:3 and 4,respectively), humIgG2 (SEQ ID NO:5), humIgG3 (SEQ ID NO:6) and humIgG4(SEQ ID NO:7) with differences between the sequences marked withasterisks.

FIG. 24 shows area under curve (AUC) for selected variants compared toanti-HER2 IgG1 (HERCEPTIN®) in a 4 hour ADCC assay. The effector cellswere PBMCs (N=5). Variant G36 (S317A; 298 in Eu numbering) with improvedbinding to FcγRIIIA showed improved ADCC activity; variant G31 (R309A;292 in Eu numbering) which did not display altered FcγRIIIA binding,also had unaltered ADCC activity; and G14 (D265A; 278 in Eu numbering)which had reduced FcγRIIIA binding, also had reduced ADCC activity.

FIGS. 25A and 25B depict binding of anti-IgE E27 IgG1 to human FcγR.FIG. 25A shows binding of E27 monomers (solid circles), dimeric (solidsquares), and hexameric (open squares) complexes to FcγRIIA-R131. FIG.25B shows binding of E27 monomers (solid circles), dimeric (solidsquares), and hexameric (open squares) complexes to FcγRIIIA-F158.Dimers were formed by mixing E27 IgG1 and a F(ab′)₂ fragment of goatanti-human k light chain at 1:0.5 molar ratio at 25° C. for 1 hr(Huizinga et al. J. Immunol. 142: 2359-2364 (1989)). Hexameric complexes(i.e. trimeric in E27 IgG1) were formed by mixing E27 IgG1 with humanIgE in a 1:1 molar ratio at 25° C. for 1 hr (Liu et al. Biochem.34:10474-10482 (1995)).

FIGS. 26A and 26B show binding sites of human IgG1 for FcγR. FIG. 26Adepicts IgG1 residues comprising the binding site for FcγRI and FcγRII.The two Fc heavy chains are in light and medium gray; carbohydrate is indark gray. Residues that affected binding to all FcγR are in red; theFcγRI binding site is comprised only of red residues. Residues thatshowed improved binding to FcγRII and FcγRIIIA are in magenta. Residuesthat showed reduced binding only to FcγRII are in yellow. Residues thatshowed ≧50% improved binding only to FcγRII are in green. FIG. 26Bdepicts IgG1 residues comprising the binding site for FcγRI andFcγRIIIA. The two Fc heavy chains are in light and medium gray;carbohydrate is in dark gray. Residues that affected binding to all FcγRare in red; the FcγRI binding site is comprised only of red residues.Residues that showed improved binding to FcγRII and FcγRIIIA are inmagenta. Residues that showed reduced binding only to FcγRIIIA are inyellow. Residues that showed ≧25% improved binding only to FcγRIIIA arein green. Glu430, involved in a salt-bridge with Lys338, is shown inblue.

FIGS. 27A, 27B and 27C show ADCC of anti-p¹⁸⁵HER2 IgG1 variants forFcγRIIIA-F158 and FcγRIIA-V158 allotypes. ADCC was performed usingp¹⁸⁵HER2-expressing SK-BR-3 cells as target and Natural Killer cells(NKs) isolated from three FcγRIIIA-Val158/Val158 donors or threeFcγRIIIA-Phe158/Phe158 donors were used as effector cells. Cytotoxicitywas detected by LDH release. Antibody-independent cell cytotoxicity(AICC) was measured using target and effector cells together (i.e. noantibody). Maximal release (MR) was measured by adding 1% Triton-100™ totarget cells. Percent cytotoxicity was calculated as (LDHrelease_(sample)/MR_(target))×100. The Effector:Target ratio was plottedversus % cytotoxicity. FIG. 27A shows representative ADCC assay for IgG1variants using NK cells from an FcγRIIIA-Val158/Val158 donor. Nativeanti-p¹⁸⁵HER2 IgG1 (solid circles), Ser298Ala (solid squares), Lys334Ala(solid triangles), Ser298Ala/Lys334Ala (open circles),Ser298Ala/Glu333Ala/Lys334Ala (open squares), AICC (open triangles).FIG. 27B shows representative ADCC assay for IgG1 variants using NKcells from an FcγRIIIA-Phe158/Phe158 donor. Native anti-p¹⁸⁵HER2 IgG1(solid circles), Ser298Ala (solid squares), Lys334Ala (solid triangles),Ser298Ala/Lys334Ala (open circles), Ser298Ala/Glu333Ala/Lys334Ala (opensquares), AICC (open triangles). FIG. 27C shows a bar plot of mean %increase in ADCC of variants compared to native anti-p¹⁸⁵HER2 IgG1.Percent increase was calculated as (% cytotoxicity_(variant)−%cytotoxicity_(native IgG1))/% cytotoxicity_(native IgG1). For eachvariant, the mean and standard deviation are for 13 data points usingthree different donors. For all variants the FcγRIIIA-Phe158/Phe158donors showed a significant increase in ADCC over the increase seen forFcγRIIIA-Val158/Val158 donors (P<0.0001 for all variants using pairedt-test).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions

Throughout the present specification and claims, the numbering of theresidues in an immunoglobulin heavy chain is that of the EU index as inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991), expressly incorporated herein by reference. The “EU index as inKabat” refers to the residue numbering of the human IgG1 EU antibody.

A “parent polypeptide” is a polypeptide comprising an amino acidsequence which lacks one or more of the Fc region modificationsdisclosed herein and which differs in effector function compared to apolypeptide variant as herein disclosed. The parent polypeptide maycomprise a native sequence Fc region or an Fc region with pre-existingamino acid sequence modifications (such as additions, deletions and/orsubstitutions).

The term “Fc region” is used to define a C-terminal region of animmunoglobulin heavy chain, e.g., as shown in FIG. 1. The “Fc region”may be a native sequence Fc region or a variant Fc region. Although theboundaries of the Fc region of an immunoglobulin heavy chain might vary,the human IgG heavy chain Fc region is usually defined to stretch froman amino acid residue at position Cys226, or from Pro230, to thecarboxyl-terminus thereof. The Fc region of an immunoglobulin generallycomprises two constant domains, CH2 and CH3, as shown, for example, inFIG. 1.

The “CH2 domain” of a human IgG FC region (also referred to as “Cγ2”domain) usually extends from about amino acid 231 to about amino acid340. The CH2 domain is unique in that it is not closely paired withanother domain. Rather, two N-linked branched carbohydrate chains areinterposed between the two CH2 domains of an intact native IgG molecule.It has been speculated that the carbohydrate may provide a substitutefor the domain-domain pairing and help stabilize the CH2 domain. Burton,Molec. Immunol. 22:161-206 (1985).

The “CH3 domain” comprises the stretch of residues C-terminal to a CH2domain in an Fc region (i.e. from about amino acid residue 341 to aboutamino acid residue 447 of an IgG)

A “functional Fc region” possesses an “effector function” of a nativesequence Fc region. Exemplary “effector functions” include C1q binding;complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor; BCR), etc.Such effector functions generally require the Fc region to be combinedwith a binding domain (e.g. an antibody variable domain) and can beassessed using various assays as herein disclosed, for example.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. Nativesequence human Fc regions are shown in FIG. 23 and include a nativesequence human IgG1 Fc region (non-A and A allotypes); native sequencehuman IgG2 Fc region; native sequence human IgG3 Fc region; and nativesequence human IgG4 Fc region as well as naturally occurring variantsthereof. Native sequence murine Fc regions are shown in FIG. 22A.

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one“amino acid modification” as herein defined. Preferably, the variant Fcregion has at least one amino acid substitution compared to a nativeSequence Fc region or to the Fc region of a parent polypeptide, e.g.from about one to about ten amino acid substitutions, and preferablyfrom about one to about five amino acid substitutions in a nativesequence Fc region or in the Fc region of the parent polypeptide. Thevariant Fc region herein will preferably possess at least about 80%homology with a native sequence Fc region and/or with an Fc region of aparent polypeptide, and most preferably at least about 90% homologytherewith, more preferably at east about 95% homology therewith.

“Homology” is defined as the percentage of residues in the amino acidsequence variant that are identical after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology.Methods and computer programs for the alignment are well known in theart. One such computer program is “Align 2”, authored by Genentech,Inc., which was filed with user documentation in the United StatesCopyright Office, Washington, D.C. 20559, on Dec. 10, 1991.

The term “Fc region-containing polypeptide” refers to a polypeptide,such as an antibody or immunoadhesin (see definitions below), whichcomprises an Fc region.

The terms “Fc receptor” or “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. The preferred FcR is a nativesequence human FcR. Moreover, a preferred FcR is one which binds an IgGantibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII,and FcγRIII subclasses, including allelic variants and alternativelyspliced forms of these receptors. FcγRII receptors include FcγRIIA (an“activating receptor”) and FcγRIIB (an “inhibiting receptor”), whichhave similar amino acid sequences that differ primarily in thecytoplasmic domains thereof. Activating receptor FcγRIIA contains animmunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmicdomain. Inhibiting receptor FcγRIIB contains an immunoreceptortyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (seereview M. in Daëron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs arereviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capelet al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin.Med. 126:330-41 (1995). Other FcRs, including those to be identified inthe future, are encompassed by the term “FcR” herein. The term alsoincludes the neonatal receptor, FcRn, which is responsible for thetransfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). The termincludes allotypes, such as FcγRIIIA allotypes: FcγRIIIA-Phe158,FcγRIIIA-Val158, FcγRIIA-R131 and/or FcγRIIA-H131.

Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFcRs (e.g. Natural Killer (NK) cells, neutrophils, and macrophages)recognize bound antibody on a target cell and subsequently cause lysisof the target cell. The primary cells for mediating ADCC, NK cells,express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils;with PBMCs and NK cells being preferred. The effector cells may beisolated from a native source thereof, e.g. from blood or PBMCs asdescribed herein.

A polypeptide variant with “altered” FcR binding affinity or ADCCactivity is one which has either enhanced or diminished FcR bindingactivity and/or ADCC activity compared to a parent polypeptide or to apolypeptide comprising a native sequence Fc region. The polypeptidevariant which “displays increased binding” to an FcR binds at least oneFcR with better affinity than the parent polypeptide. The improvement inbinding compared to a parent polypeptide may be about 25% or betterimprovement, e.g. up to about 200% or about 1000% improvement inbinding. The polypeptide variant which “displays decreased binding” toan FcR, binds at least one FcR with worse affinity than a parentpolypeptide. The decrease in binding compared to a parent polypeptidemay be about 40% or more decrease in binding, e.g. down to a variantwhich possess little or no appreciable binding to the FcR. Such variantswhich display decreased binding to an FcR may possess little or noappreciable binding to an FcR, e.g., 0-20% binding to the FcR comparedto a native sequence IgG Fc region, e.g. as determined in the Examplesherein.

The polypeptide variant which binds an FcR with “better affinity” than aparent polypeptide, is one which binds any one or more of the aboveidentified FcRs with substantially better binding affinity than theparent antibody, when the amounts of polypeptide variant and parentpolypeptide in the binding assay are essentially the same. For example,the polypeptide variant with improved FcR binding affinity may displayfrom about 1.15 fold to about 100 fold, e.g. from about 1.2 fold toabout 50 fold improvement in FcR binding affinity compared to the parentpolypeptide, where FcR binding affinity is determined, for example, asdisclosed in the Examples herein.

The polypeptide variant which “mediates antibody-dependent cell-mediatedcytotoxicity (ADCC) in the presence of human effector cells moreeffectively” than a parent antibody is one which in vitro or in vivo issubstantially more effective at mediating ADCC, when the amounts ofpolypeptide variant and parent antibody used in the assay areessentially the same. Generally, such variants will be identified usingthe in vitro ADCC assay as herein disclosed, but other assays or methodsfor determining ADCC activity, e.g. in an animal model etc, arecontemplated. The preferred variant is from about 1.5 fold to about 100fold, e.g. from about two fold to about fifty fold, more effective atmediating ADCC than the parent, e.g. in the in vitro assay disclosedherein.

An “amino acid modification” refers to a change in the amino acidsequence of a predetermined amino acid sequence. Exemplary modificationsinclude an amino acid substitution, insertion and/or deletion. Thepreferred amino acid modification herein is a substitution.

An “amino acid modification at” a specified position, e.g. of the Fcregion, refers to the substitution or deletion of the specified residue,or the insertion of at least one amino acid residue adjacent thespecified residue. By insertion “adjacent” a specified residue is meantinsertion within one to two residues thereof. The insertion may beN-terminal or C-terminal to the specified residue.

An “amino acid substitution” refers to the replacement of at least oneexisting amino acid residue in a predetermined amino acid sequence withanother different “replacement” amino acid residue. The replacementresidue or residues may be “naturally occurring amino acid residues”(i.e. encoded by the genetic code) and selected from the groupconsisting of: alanine (Ala); arginine (Arg); asparagine (Asn); asparticacid (Asp); cysteine (Cys); glutamine (Gln); glutamic acid (Glu);glycine (Gly); histidine (His); isoleucine (Ile): leucine (Leu); lysine(Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine(Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine(Val). Preferably, the replacement residue is not cysteine. Substitutionwith one or more non-naturally occurring amino acid residues is alsoencompassed by the definition of an amino acid substitution herein. A“non-naturally occurring amino acid residue” refers to a residue, otherthan those naturally occurring amino acid residues listed above, whichis able to covalently bind adjacent amino acid residues(s) in apolypeptide chain. Examples of non-naturally occurring amino acidresidues include norleucine, ornithine, norvaline, homoserine and otheramino acid residue analogues such as those described in Ellman et al.Meth. Enzym. 202:301-336 (1991). To generate such non-naturallyoccurring amino acid residues, the procedures of Noren et al. Science244:182 (1989) and Ellman et al., supra, can be used. Briefly, theseprocedures involve chemically activating a suppressor tRNA with anon-naturally occurring amino acid residue followed by in vitrotranscription and translation of the RNA.

An “amino acid insertion” refers to the incorporation of at least oneamino acid into a predetermined amino acid sequence. While the insertionwill usually consist of the insertion of one or two amino acid residues,the present application contemplates larger “peptide insertions”, e.g.insertion of about three to about five or even up to about ten aminoacid residues. The inserted residue(s) may be naturally occurring ornon-naturally occurring as disclosed above.

An “amino acid deletion” refers to the removal of at least one aminoacid residue from a predetermined amino acid sequence.

“Hinge region” is generally defined as stretching from Glu216 to Pro230of human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). Hinge regionsof other IgG isotypes may be aligned with the IgG1 sequence by placingthe first and last cysteine residues forming inter-heavy chain S—S bondsin the same positions.

The “lower hinge region” of an Fc region is normally defined as thestretch of residues immediately C-terminal to the hinge region, i.e.residues 233 to 239 of the Fc region. Prior to the present invention,FcγR binding was generally attributed to amino acid residues in thelower hinge region of an IgG Fc region.

“C1q” is a polypeptide that includes a binding site for the Fc region ofan immunoglobulin. C1q together with two serine proteases, C1r and C1s,forms the complex C1, the first component of the complement dependentcytotoxicity (CDC) pathway. Human C1q can be purchased commerciallyfrom, e.g. Quidel, San Diego, Calif.

The term “binding domain” refers to the region of a polypeptide thatbinds to another molecule. In the case of an FcR, the binding domain cancomprise a portion of a polypeptide chain thereof (e.g. the a chainthereof) which is responsible for binding an Fc region. One usefulbinding domain is the extracellular domain of an FcR α chain.

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired biological activity.

“Antibody fragments”, as defined for the purpose of the presentinvention, comprise a portion of an intact antibody, generally includingthe antigen binding or variable region of the intact antibody or the Fcregion of an antibody which retains FcR binding capability. Examples ofantibody fragments include linear antibodies; single-chain antibodymolecules; and multispecific antibodies formed from antibody fragments.The antibody fragments preferably retain at least part of the hinge andoptionally the CH1 region of an IgG heavy chain. More preferably, theantibody fragments retain the entire constant region of an IgG heavychain, and include an IgG light chain.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations that typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., Nature256:495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol.Biol. 222:581-597 (1991), for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin sequence. Thehumanized antibody optionally also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (i.e. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain;Kabat et al., Sequences of Proteins of immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991)) and/or those residues from a “hypervariable loop” (i.e. residues26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domainand 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). “Framework”or “FR” residues are those variable domain residues other than thehypervariable region residues as herein defined.

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the “binding domain” of a heterologous “adhesin”protein (e.g. a receptor, ligand or enzyme) with an immunoglobulinconstant domain. Structurally, the immunoadhesins comprise a fusion ofthe adhesin amino acid sequence with the desired binding specificitywhich is other than the antigen recognition and binding site (antigencombining site) of an antibody (i.e. is “heterologous”) and animmunoglobulin constant domain sequence.

The term “ligand binding domain” as used herein refers to any nativecell-surface receptor or any region or derivative thereof retaining atleast a qualitative ligand binding ability of a corresponding nativereceptor. In a specific embodiment, the receptor is from a cell-surfacepolypeptide having an extracellular domain that is homologous to amember of the immunoglobulin supergenefamily. Other receptors, which arenot members of the immunoglobulin supergenefamily but are nonethelessspecifically covered by this definition, are receptors for cytokines,and in particular receptors with tyrosine kinase activity (receptortyrosine kinases), members of the hematopoietin and nerve growth factorreceptor superfamilies, and cell adhesion molecules, e.g. (E-, L- andP-) selectins.

The term “receptor binding domain” is used to designate any nativeligand for a receptor, including cell adhesion molecules, or any regionor derivative of such native ligand retaining at least a qualitativereceptor binding ability of a corresponding native ligand. Thisdefinition, among others, specifically includes binding sequences fromligands for the above-mentioned receptors.

An “antibody-immunoadhesin chimera” comprises a molecule that combinesat least one binding domain of an antibody (as herein defined) with atleast one immunoadhesin (as defined in this application). Exemplaryantibody-immunoadhesin chimeras are the bispecific CD4-IgG chimerasdescribed in Berg et al., PNAS (USA) 88:4723-4727 (1991) and Chamow etal., J. Immunol. 153:4268 (1994).

An “isolated” polypeptide is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the polypeptide,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the polypeptide willbe purified (1) to greater than 95% by weight of polypeptide asdetermined by the Lowry method, and most preferably more than 99% byweight, (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under reducing ornonreducing conditions using Coomassie blue or, preferably, silverstain. Isolated polypeptide includes the polypeptide in situ withinrecombinant cells since at least one component of the polypeptide'snatural environment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented.

A “disorder” is any condition that would benefit from treatment with thepolypeptide variant. This includes chronic and acute disorders ordiseases including those pathological conditions which predispose themammal to the disorder in question. In one embodiment, the disorder iscancer.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidneycancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma and various types of head and neck cancer.

A “HER2-expressing cancer” is one comprising cells which have HER2receptor protein (Semba et al., PNAS (USA) 82:6497-6501 (1985) andYamamoto et al. Nature 319:230-234 (1986) (Genebank accession numberX03363)) present at their cell surface, such that an anti-HER2 antibodyis able to bind to the cancer.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to thepolypeptide. The label may be itself be detectable (e.g., radioisotopelabels or fluorescent labels) or, in the case of an enzymatic label, maycatalyze chemical alteration of a substrate compound or compositionwhich is detectable.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe polypeptide nucleic acid. An isolated nucleic acid molecule is otherthan in the form or setting in which it is found in nature. Isolatednucleic acid molecules therefore are distinguished from the nucleic acidmolecule as it exists in natural cells. However, an isolated nucleicacid molecule includes a nucleic acid molecule contained in cells thatordinarily express the polypeptide where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The expression “control sequences” refers to DNA sequences necessary forthe expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

The term “molecular complex” when used herein refers to the relativelystable structure which forms when two or more heterologous molecules(e.g. polypeptides) bind (preferably noncovalently) to one another. Thepreferred molecular complex herein is an immune complex.

“Immune complex” refers to the relatively stable structure which formswhen at least one target molecule and at least one heterologous Fcregion-containing polypeptide bind to one another forming a largermolecular weight complex. Examples of immune complexes areantigen-antibody aggregates and target molecule-immunoadhesinaggregates. The term “immune complex” as used herein, unless indicatedotherwise, refers to an ex vivo complex (i.e. other than the form orsetting in which it may be found in nature). However, the immune complexmay be administered to a mammal, e.g. to evaluate clearance of theimmune complex in the mammal.

The term “target molecule” refers to a molecule, usually a polypeptide,which is capable of being bound by a heterologous molecule and has oneor more binding sites for the heterologous molecule. The term “bindingsite” refers to a region of a molecule to which another molecule canbind. The “first target molecule” herein comprises at least two distinctbinding sites (for example, two to five separate binding sites) for ananalyte (e.g. an Fc region-containing polypeptide) such that at leasttwo analyte molecules can bind to the first target molecule. In thepreferred embodiment of the invention, the two or more binding sites areidentical (e.g. having the same amino acid sequence, where the targetmolecule is a polypeptide). In Example 1 below, the first targetmolecule was IgE and had two separate binding sites in the Fc regionthereof to which the Fc region-containing polypeptide (an anti-IgEantibody, E27) could bind. Other first target molecules include dimersof substantially identical monomers (e.g. neurotrophins, IL8 and VEGF)or are polypeptides comprising two or more substantially identicalpolypeptide chains (e.g. antibodies or immunoadhesins). The “secondtarget molecule” comprises at least two distinct binding sites (forexample, two to five separate binding sites) for the first targetmolecule such that at least two first target molecules can bind to thesecond target molecule. Preferably, the two or more binding sites areidentical (e.g. having the same amino acid sequence, where the targetmolecule is a polypeptide). In Example 2, the second target molecule wasVEGF, which has a pair of distinct binding sites to which the variabledomain of the IgE antibody could bind. Other second target molecules arecontemplated, e.g. other dimers of substantially identical monomers(e.g. neurotrophins or IL8) or polypeptides comprising two or moresubstantially identical domains (e.g. antibodies or immunoadhesins).

An “analyte” is a substance that is to be analyzed. The preferredanalyte is an Fc region-containing polypeptide that is to be analyzedfor its ability to bind to an Fc receptor.

A “receptor” is a polypeptide capable of binding at least one ligand.The preferred receptor is a cell-surface receptor having anextracellular ligand-binding domain and, optionally, other domains (e.g.transmembrane domain, intracellular domain and/or membrane anchor). Thereceptor to be evaluated in the assay described herein may be an intactreceptor or a fragment or derivative thereof (e.g. a fusion proteincomprising the binding domain of the receptor fused to one or moreheterologous polypeptides). Moreover, the receptor to be evaluated forits binding properties may be present in a cell or isolated andoptionally coated on an assay plate or some other solid phase.

The phrase “low affinity receptor” denotes a receptor that has a weakbinding affinity for a ligand of interest, e.g. having a bindingconstant of about 50 nM or worse affinity. Exemplary low affinityreceptors include FcγRII and FcγRIII.

II. Modes for Carrying Out the Invention

The invention herein relates to a method for making a polypeptidevariant. The “parent”, “starting” or “nonvariant” polypeptide isprepared using techniques available in the art for generatingpolypeptides comprising an Fc region. In the preferred embodiment of theinvention, the parent polypeptide is an antibody and exemplary methodsfor generating antibodies are described in more detail in the followingsections. The parent polypeptide may, however, be any other polypeptidecomprising an Fc region, e.g. an immunoadhesin. Methods for makingimmunoadhesins are elaborated in more detail hereinbelow.

In an alternative embodiment, a variant Fc region may be generatedaccording to the methods herein disclosed and this “variant Fc region”can be fused to a heterologous polypeptide of choice, such as anantibody variable domain or binding domain of a receptor or ligand.

The parent polypeptide comprises an Fc region. Generally the Fc regionof the parent polypeptide will comprise a native sequence Fc region, andpreferably a human native sequence Fc region. However, the Fc region ofthe parent polypeptide may have one or more pre-existing amino acidsequence alterations or modifications from a native sequence Fc region.For example, the C1q binding activity of the Fc region may have beenpreviously altered (other types of Fc region modifications are describedin more detail below). In a further embodiment the parent polypeptide Fcregion is “conceptual” and, while it does not physically exist, theantibody engineer may decide upon a desired variant Fc region amino acidsequence and generate a polypeptide comprising that sequence or a DNAencoding the desired variant Fc region amino acid sequence.

In the preferred embodiment of the invention, however, a nucleic acidencoding an Fc region of a parent polypeptide is available and thisnucleic acid sequence is altered to generate a variant nucleic acidsequence encoding the Fc region variant.

DNA encoding an amino acid sequence variant of the starting polypeptideis prepared by a variety of methods known in the art. These methodsinclude, but are not limited to, preparation by site-directed (oroligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassettemutagenesis of an earlier prepared DNA encoding the polypeptide

Site-directed mutagenesis is a preferred method for preparingsubstitution variants. This technique is well known in the art (see,e.g., Carter et al. Nucleic Acids Res. 13:4431-4443 (1985) and Kunkel etal., Proc. Natl. Acad. Sci. USA 82:488 (1987)). Briefly, in carrying outsite-directed mutagenesis of DNA, the starting DNA is altered by firsthybridizing an oligonucleotide encoding the desired mutation to a singlestrand of such starting DNA. After hybridization, a DNA polymerase isused to synthesize an entire second strand, using the hybridizedoligonucleotide as a primer, and using the single strand of the startingDNA as a template. Thus, the oligonucleotide encoding the desiredmutation is incorporated in the resulting double-stranded DNA.

PCR mutagenesis is also suitable for making amino acid sequence variantsof the starting polypeptide. See Higuchi, in PCR Protocols, pp. 177-183(Academic Press, 1990); and Vallette et al., Nuc. Acids Res. 17:723-733(1989). Briefly, when small amounts of template DNA are used as startingmaterial in a PCR, primers that differ slightly in sequence from thecorresponding region in a template DNA can be used to generaterelatively large quantities of a specific DNA fragment that differs fromthe template sequence only at the positions where the primers differfrom the template.

Another method for preparing variants, cassette mutagenesis, is based onthe technique described by Wells et al., Gene 34:315-323 (1985). Thestarting material is the plasmid (or other vector) comprising thestarting polypeptide DNA to be mutated. The codon(s) in the starting DNAto be mutated are identified. There must be a unique restrictionendonuclease site on each side of the identified mutation site(s). If nosuch restriction sites exist, they may be generated using theabove-described oligonucleotide-mediated mutagenesis method to introducethem at appropriate locations in the starting polypeptide DNA. Theplasmid DNA is cut at these sites to linearize it. A double-strandedoligonucleotide encoding the sequence of the DNA between the restrictionsites but containing the desired mutation(s) is synthesized usingstandard procedures, wherein the two strands of the oligonucleotide aresynthesized separately and then hybridized together using standardtechniques. This double-stranded oligonucleotide is referred to as thecassette. This cassette is designed to have 5′ and 3′ ends that arecompatible with the ends of the linearized plasmid, such that it can bedirectly ligated to the plasmid. This plasmid now contains the mutatedDNA sequence.

Alternatively, or additionally, the desired amino acid sequence encodinga polypeptide variant can be determined, and a nucleic acid sequenceencoding such amino acid sequence variant can be generatedsynthetically.

The amino acid sequence of the parent polypeptide is modified in orderto generate a variant Fc region with altered Fc receptor bindingaffinity or activity in vitro and/or in vivo and/or alteredantibody-dependent cell-mediated cytotoxicity (ADCC) activity in vitroand/or in vivo.

Generally, the modification entails one or more amino acidsubstitutions. In one embodiment, the replacement residue does notcorrespond to a residue present in the same position in any of thenative sequence Fc regions in FIG. 22A. For example, according to thisembodiment of the invention, Pro331 of a human IgG3 or IgG1 Fc region isreplaced with a residue other than Ser (the corresponding alignedresidue found in native sequence human IgG4). In one embodiment, theresidue in the parent polypeptide which is substituted with areplacement residue is not an alanine and/or is not residue Ala339 of anFc region. In the case of an amino acid substitution, preferably theresidue in the parent polypeptide is replaced with an alanine residue.However, the present invention contemplates replacement of the residueof the parent polypeptide with any other amino acid residue. Thesubstitution may, for example, be a “conservative substitution”. Suchconservative substitutions are shown in Table 1 under the heading of“preferred substitution”. More substantial changes may be achieved bymaking one or more “exemplary substitutions” which are not the preferredsubstitution in Table 1.

TABLE 1 Exemplary Preferred Original Residue Substitutions SubstitutionAla (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his;lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) aspasp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu;val; met; ala; leu phe; norleucine Leu (L) norleucine; ile; val; ilemet; ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe(F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T)ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile;leu; met; phe; leu ala; norleucine

Substantial modifications in the biological properties of the Fc regionmay be accomplished by selecting substitutions that differ significantlyin their effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gin, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for a member of another class. Conservative andnon-conservative amino acid substitutions are exemplified in Table 7hereinbelow.

As is demonstrated in Example 4 herein, one can engineer an Fc regionvariant with altered binding affinity for one or more FcRs. As was shownin that Example, different classes of Fc region variants can be madee.g., as summarized in the following table. Where the variant Fc regionhas more than one amino acid substitution, generally, but notnecessarily, amino acid substitutions in the same class are combined toachieve the desired result.

TABLE 2 CLASSES OF Fc REGION VARIANTS Class FcR binding propertyPosition of Fc region substitution(s) 1 reduced binding to all FcγR233-236#, 238, 265#, 297#*, 327 G1n, 329 2 reduced binding to bothFcγRII and 270, 295, 327Ser FcγRIII 3 improved binding to both FcγRIIand 256, 290 FcγRIII 4 improved binding to FcγRII and no 255, 258, 267,272, 276, 280, 285, 286, 307, effect on FcγRIII binding 309, 315, 326,331, 337, 378, 430 5 improved binding to FcγRII and 268, 301, 322reduced binding to FcγRIII 6 reduced binding to FcγRII and no 292, 414effect on FcγyRIII binding 7 reduced binding to FcγRII and 298 improvedbinding to FcγRIII 8 no effect on FcγRII binding and 239, 269, 293, 296,303, 327Gly, 338, 376 reduced binding to FcγRIII 9 no effect on FcγRIIbinding and 333, 334, 339# improved binding to FcγRIII 10 effect onlyFcRn 253, 254, 288, 305, 311, 312, 317, 360, 362, 380, 382, 415, 424,433, 434, 435, 436 *deglycosylated version #Preferably combined withother Fc modification(s), (e.g. as disclosed herein)

Aside from amino acid substitutions, the present invention contemplatesother modifications of the parent Fc region amino acid sequence in orderto generate an Fc region variant with altered effector function.

One may, for example, delete one or more amino acid residues of the Fcregion in order to reduce binding to an FcR. Generally, one will deleteone or more of the Fc region residues identified herein as effecting FcRbinding (see Example 4 below) in order to generate such an Fc regionvariant. Generally, no more than one to about ten Fc region residueswill be deleted according to this embodiment of the invention. The Fcregion herein comprising one or more amino acid deletions willpreferably retain at least about 80%, and preferably at least about 90%,and most preferably at least about 95%, of the parent Fc region or of anative sequence human Fc region.

One may also make amino acid insertion Fc region variants, whichvariants have altered effector function. For example, one may introduceat least one amino acid residue (e.g. one to two amino acid residues andgenerally no more than ten residues) adjacent to one or more of the Fcregion positions identified herein as impacting FcR binding. By“adjacent” is meant within one to two amino acid residues of a Fc regionresidue identified herein. Such Fc region variants may display enhancedor diminished FcR binding and/or ADCC activity. In order to generatesuch insertion variants, one may evaluate a co-crystal structure of apolypeptide comprising a binding region of an FcR (e.g. theextracellular domain of the FcR of interest) and the Fc region intowhich the amino acid residue(s) are to be inserted (see, for example,Deisenhofer, Biochemistry 20(9):2361-2370 (1981); and Burmeister et al.,Nature 372:379-383, (1994)) in order to rationally design an Fc regionvariant with, e.g., improved FcR binding ability. Such insertion(s) willgenerally be made in an Fc region loop, but not in the secondarystructure (i.e. in a β-strand) of the Fc region.

By introducing the appropriate amino acid sequence modifications in aparent Fc region, one can generate a variant Fc region which (a)mediates antibody-dependent cell-mediated cytotoxicity (ADCC) in thepresence of human effector cells more effectively and/or (b) binds an Fcgamma receptor (FcγR) with better affinity than the parent polypeptide.Such Fc region variants will generally comprise at least one amino acidmodification in the Fc region. Combining amino acid modifications isthought to be particularly desirable. For example, the variant Fc regionmay include two, three, four, five, etc substitutions therein, e.g. ofthe specific Fc region positions identified herein.

Preferably, the parent polypeptide Fc region is a human Fc region, e.g.a native sequence human Fc region human IgG1 (A and non-A allotypes),IgG2, IgG3 or IgG4 Fc region. Such sequences are shown in FIG. 23.

To generate an Fc region with improved ADCC activity, the parentpolypeptide preferably has pre-existing ADCC activity, e.g., itcomprises a human IgG1 or human IgG3 Fc region. In one embodiment, thevariant with improved ADCC mediates ADCC substantially more effectivelythan an antibody with a native sequence IgG1 or IgG3 Fc region and theantigen-binding region of the variant. Preferably, the variantcomprises, or consists essentially of, substitutions of two or three orall of the residues at positions 298, 333, 334 and 339 of the Fc region.Most preferably, residues at positions 298, 333, 334 and 339 aresubstituted, (e.g. with alanine residues). At position 298, thepreferred replacement residue is an ala or gly, and most preferably ala.The preferred replacement residue at position 333 is ala or asp, andmost preferably ala. The residue at position 334 is preferably replacedby one of the following amino acids: ala, gln, glu, met, tyr, his, val,leu, asn, ser, trp, with ala or glu being the most preferred replacementresidues at this position. The preferred replacement residue at position339 is a thr. Moreover, in order to generate the Fc region variant withimproved ADCC activity, one will generally engineer an Fc region variantwith improved binding affinity for FcγRIII, which is thought to be animportant FcR for mediating ADCC. For example, one may introduce anamino acid modification (e.g. a substitution) into the parent Fc regionat any one or more of amino acid positions 256, 290, 298, 312, 326, 330,333, 334, 360, 378 or 430 to generate such a variant. The variant withimproved binding affinity for FcγRIII may further have reduced bindingaffinity for FcγRII, especially reduced affinity for the inhibitingFcγRIIB receptor.

The amino acid modification(s) are preferably introduced into the CH2domain of a Fc region, since the experiments herein indicate that theCH2 domain is important for FcR binding activity. Moreover, unlike theteachings of the above-cited art, the instant application contemplatesthe introduction of a modification into a part of the Fc region otherthan in the lower hinge region thereof.

Useful amino acid positions for modification in order to generate avariant IgG Fc region with altered Fc gamma receptor (FcγR) bindingaffinity or activity include any one or more of amino acid positions238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270,272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296,298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329,330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388,389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439 of the Fcregion. Preferably, the parent Fc region used as the template togenerate such variants comprises a human IgG Fc region. Where residue331 is substituted, the parent Fc region is preferably not human nativesequence IgG3, or the variant Fc region comprising a substitution atposition 331 preferably displays increased FcR binding, e.g. to FcγRII.

To generate an Fc region variant with reduced binding to the FcγR onemay introduce an amino acid modification at any one or more of aminoacid positions 238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272,278, 289, 292, 293, 294, 295, 296, 298, 301, 303, 322, 324, 327, 329,333, 335, 338, 340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435,437, 438 or 439 of the Fc region.

Variants which display reduced binding to FcγRI, include thosecomprising an Fc region amino acid modification at any one or more ofamino acid positions 238, 265, 269, 270, 327 or 329.

Variants which display reduced binding to FcγRII include thosecomprising an Fc region amino acid modification at any one or more ofamino acid positions 238, 265, 269, 270, 292, 294, 295, 298, 303, 324,327, 329, 333, 335, 338, 373, 376, 414, 416, 419, 435, 438 or 439.

Fc region variants which display reduced binding to FcγRIII includethose comprising an Fc region amino acid modification at any one or moreof amino acid positions 238, 239, 248, 249, 252, 254, 265, 268, 269,270, 272, 278, 289, 293, 294, 295, 296, 301, 303, 322, 327, 329, 338,340, 373, 376, 382, 388, 389, 416, 434, 435 or 437.

Variants with improved binding to one or more FcγRs may also be made.Such Fc region variants may comprise an amino acid modification at anyone or more of amino acid positions 255, 256, 258, 267, 268, 272, 276,280, 283, 285, 286, 290, 298, 301, 305, 307, 309, 312, 315, 320, 322,326, 330, 331, 333; 334, 337, 340, 360, 378, 398 or 430 of the Fcregion.

For example, the variant with improved FcγR binding activity may displayincreased binding to FcγRIII, and optionally may further displaydecreased binding to FcγRII; e.g. the variant may comprise an amino acidmodification at position 298 and/or 333 of an Fc region.

Variants with increased binding to FcγRII include those comprising anamino acid modification at any one or more of amino acid positions 255,256, 258, 267, 268, 272, 276, 280, 283, 285, 286, 290, 301, 305, 307,309, 312, 315, 320, 322, 326, 330, 331, 337, 340, 378, 398 or 430 of anFc region. Such variants may further display decreased binding toFcγRIII. For example, they may include an Fc region amino acidmodification at any one or more of amino acid positions 268, 272, 298,301, 322 or 340.

While it is preferred to alter binding to a FcγR, Fc region variantswith altered binding affinity for the neonatal receptor (FcRn) are alsocontemplated herein. Fc region variants with improved affinity for FcRnare anticipated to have longer serum half-lives, and such molecules willhave useful applications in methods of treating mammals where longhalf-life of the administered polypeptide is desired, e.g., to treat achronic disease or disorder. Fc region variants with decreased FcRnbinding affinity, on the contrary, are expected to have shorterhalf-lives, and such molecules may, for example, be administered to amammal where a shortened circulation time may be advantageous, e.g. forin vivo diagnostic imaging or for polypeptides which have toxic sideeffects when left circulating in the blood stream for extended periods,etc. Fc region variants with decreased FcRn binding affinity areanticipated to be less likely to cross the placenta, and thus may beutilized in the treatment of diseases or disorders in pregnant women.

Fc region variants with altered binding affinity for FcRn include thosecomprising an Fc region amino acid modification at any one or more ofamino acid positions 238, 252, 253, 254, 255, 256, 265, 272, 286, 288,303, 305, 307, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380,382, 386, 388, 400, 413, 415, 424, 433, 434, 435, 436, 439 or 447. Thosewhich display reduced binding to FcRn will generally comprise an Fcregion amino acid modification at any one or more of amino acidpositions 252, 253, 254, 255, 288, 309, 386, 388, 400, 415, 433, 435,436, 439, or 447; and those with increased binding to FcRn will usuallycomprise an Fc region amino acid modification at any one or more ofamino acid positions 238, 256, 265, 272, 286, 303, 305, 307, 311, 312,317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434.

The polypeptide variant(s) prepared as described above may be subjectedto further modifications, oftentimes depending on the intended use ofthe polypeptide. Such modifications may involve further alteration ofthe amino acid sequence (substitution, insertion and/or deletion ofamino acid residues), fusion to heterologous polypeptide(s) and/orcovalent modifications. Such “further modifications” may be made priorto, simultaneously with, or following, the amino acid modification(s)disclosed above which result in an alteration of Fc receptor bindingand/or ADCC activity. In one embodiment, one may combine the Fc regionmodification herein with Fc region substitutions disclosed in thereferences cited in the “Related Art” section of this application.

Alternatively or additionally, it may be useful to combine the aboveamino acid modifications with one or more further amino acidmodifications that alter FcRn binding and/or half-life of the antibodyand/or C1q binding and/or complement dependent cytotoxicity function ofthe Fc region.

The starting polypeptide of particular interest herein is usually onethat binds to C1q and displays complement dependent cytotoxicity (CDC).The further amino acid substitutions described herein will generallyserve to alter the ability of the starting polypeptide to bind to C1qand/or modify its complement dependent cytotoxicity function, e.g. toreduce and preferably abolish these effector functions. However,polypeptides comprising substitutions at one or more of the describedpositions with improved C1q binding and/or complement dependentcytotoxicity (CDC) function are contemplated herein. For example, thestarting polypeptide may be unable to bind C1q and/or mediate CDC andmay be modified according to the teachings herein such that it acquiresthese further effector functions. Moreover, polypeptides withpre-existing C1q binding activity, optionally further having the abilityto mediate CDC may be modified such that one or both of these activitiesare enhanced.

To generate an Fc region with altered C1q binding and/or complementdependent cytotoxicity (CDC) function, the amino acid positions to bemodified are generally selected from heavy chain positions 270, 322,326, 327, 329, 331, 333, and 334, where the numbering of the residues inan IgG heavy chain is that of the EU index as in Kabat et al., Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991). In one embodiment,only one of the eight above-identified positions is altered in order togenerate the polypeptide variant region with altered C1q binding and/orcomplement dependent cytotoxicity (CDC) function. Preferably onlyresidue 270, 329 or 322 is altered if this is the case. Alternatively,two or more of the above-identified positions are modified. Ifsubstitutions are to be combined, generally substitutions which enhancehuman C1q binding (e.g. at residue positions 326, 327, 333 and 334) orthose which diminish human C1q binding (e.g., at residue positions 270,322, 329 and 331) are combined. In the latter embodiment, all fourpositions (i.e., 270, 322, 329 and 331) may be substituted. Preferably,further substitutions at two, three or all of positions 326, 327, 333 or334 are combined, optionally with other Fc region substitutions, togenerate a polypeptide with improved human C1q binding and preferablyimproved CDC activity in vitro or in vivo.

Proline is conserved at position 329 in human IgG's. This residue ispreferably replaced with alanine, however substitution with any otheramino acid is contemplated, e.g., serine, threonine, asparagine, glycineor valine.

Proline is conserved at position 331 in human IgG1, IgG2 and IgG3, butnot IgG4 (which has a serine residue at position 331). Residue 331 ispreferably replaced by alanine or another amino acid, e.g. serine (forIgG regions other than IgG4), glycine or valine.

Lysine 322 is conserved in human IgGs, and this residue is preferablyreplaced by an alanine residue, but substitution with any other aminoacid residue is contemplated, e.g. serine, threonine, glycine or valine.

D270 is conserved in human IgGs, and this residue may be replaced byanother amino acid residue, e.g. alanine, serine, threonine, glycine,valine, or lysine.

K326 is also conserved in human IgGs. This residue may be substitutedwith another residue including, but not limited to, valine, glutamicacid, alanine, glycine, aspartic acid, methionine or tryptophan, withtryptophan being preferred.

Likewise, E333 is also conserved in human IgGs. E333 is preferablyreplaced by an amino acid residue with a smaller side chain volume, suchas valine, glycine, alanine or serine, with serine being preferred.

K334 is conserved in human IgGs and may be substituted with anotherresidue such as alanine or other residue.

In human IgG1 and IgG3, residue 327 is an alanine. In order to generatea variant with improved C1q binding, this alanine may be substitutedwith another residue such as glycine. In IgG2 and IgG4, residue 327 is aglycine and this may be replaced by alanine (or another residue) todiminish C1q binding.

As disclosed above, one can design an Fc region with altered effectorfunction, e.g., by modifying C1q binding and/or FcR binding and therebychanging CDC activity and/or ADCC activity. For example, one cangenerate a variant Fc region with improved C1q binding and improvedFcγRIII binding; e.g. having both improved ADCC activity and improvedCDC activity. Alternatively, where one desires that effector function bereduced or ablated, one may engineer a variant Fc region with reducedCDC activity and/or reduced ADCC activity. In other embodiments, one mayincrease only one of these activities, and optionally also reduce theother activity, e.g. to generate an Fc region variant with improved ADCCactivity, but reduced CDC activity and vice versa.

With respect to further amino acid sequence alterations, any cysteineresidue not involved in maintaining the proper conformation of thepolypeptide variant also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcross linking.

Another type of amino acid substitution serves to alter theglycosylation pattern of the polypeptide. This may be achieved bydeleting one or more carbohydrate moieties found in the polypeptide,and/or adding one or more glycosylation sites that are not present inthe polypeptide. Glycosylation of polypeptides is typically eitherN-linked or O-linked. N-linked refers to the attachment of thecarbohydrate moiety to the side chain of an asparagine residue. Thetripeptide sequences asparagine-X-serine and asparagine-X-threonine,where X is any amino acid except proline, are the recognition sequencesfor enzymatic attachment of the carbohydrate moiety to the asparagineside chain. Thus, the presence of either of these tripeptide sequencesin a polypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used. Addition of glycosylation sites to thepolypeptide is conveniently accomplished by altering the amino acidsequence such that it contains one or more of the above-describedtripeptide sequences (for N-linked glycosylation sites). The alterationmay also be made by the addition of, or substitution by, one or moreserine or threonine residues to the sequence of the original polypeptide(for O-linked glycosylation sites). An exemplary glycosylation varianthas an amino acid substitution of residue Asn 297 of the heavy chain.

Moreover, the class, subclass or allotype of the Fc region may bealtered by one or more further amino acid substitutions to generate anFc region with an amino acid sequence more homologous to a differentclass, subclass or allotype as desired. For example, a murine Fc regionmay be altered to generate an amino acid sequence more homologous to ahuman Fc region; a human non-A allotype IgG1 Fc region may be modifiedto achieve a human A allotype IgG1 Fc region etc. In one embodiment, theamino modification(s) herein which alter FcR binding and/or ADCCactivity are made in the CH2 domain of the Fc region and the CH3 domainis deleted or replaced with another dimerization domain. Preferably,however, the CH3 domain is retained (aside from amino acid modificationstherein which alter effector function as herein disclosed).

The polypeptide variant may be subjected to one or more assays toevaluate any change in biological activity compared to the startingpolypeptide.

Preferably the polypeptide variant essentially retains the ability tobind antigen compared to the nonvariant polypeptide, i.e. the bindingcapability is no worse than about 20 fold, e.g. no worse than about 5fold of that of the nonvariant polypeptide. The binding capability ofthe polypeptide variant may be determined using techniques such asfluorescence activated cell sorting (FACS) analysis orradioimmunoprecipitation (RIA), for example.

The ability of the polypeptide variant to bind an FcR may be evaluated.Where the FcR is a high affinity Fc receptor, such as FcγRI, FcRn orFcγRIIIA-V158, binding can be measured by titrating monomericpolypeptide variant and measuring bound polypeptide variant using anantibody which specifically binds to the polypeptide variant in astandard ELISA format (see Example 2 below). Another FcR binding assayfor low affinity FcRs is described in Examples 1 and 4.

To assess ADCC activity of the polypeptide variant, an in vitro ADCCassay, such as that described in Example 4 may be performed usingvarying effector:target ratios. Useful “effector cells” for such assaysinclude peripheral blood mononuclear cells (PBMC) and Natural Killer(NK) cells. Alternatively, or additionally, ADCC activity of thepolypeptide variant may be assessed in vivo, e.g., in a animal modelsuch as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

The ability of the variant to bind C1q and mediate complement dependentcytotoxicity (CDC) may be assessed.

To determine C1q binding, a C1q binding ELISA may be performed. Briefly,assay plates may be coated overnight at 4° C. with polypeptide variantor starting polypeptide (control) in coating buffer. The plates may thenbe washed and blocked. Following washing, an aliquot of human C1q may beadded to each well and incubated for 2 hrs at room temperature.Following a further wash, 100 μl of a sheep anti-complement C1qperoxidase conjugated antibody may be added to each well and incubatedfor 1 hour at room temperature. The plate may again be washed with washbuffer and 100 μl of substrate buffer containing OPD (O-phenylenediaminedihydrochloride (Sigma)) may be added to each well. The oxidationreaction, observed by the appearance of a yellow color, may be allowedto proceed for 30 minutes and stopped by the addition of 100 μl of 4.5 NH₂SO₄. The absorbance may then read at (492-405) nm.

An exemplary polypeptide variant is one that displays a “significantreduction in C1q binding” in this assay. This means that about 100 μg/mlof the polypeptide variant displays about 50 fold or more reduction inC1q binding compared to 100 μg/ml of a control antibody having anonmutated IgG1 Fc region. In the most preferred embodiment, thepolypeptide variant “does not bind C1q”, i.e. 100 μg/ml of thepolypeptide variant displays about 100 fold or more reduction in C1qbinding compared to 100 μg/ml of the control antibody.

Another exemplary variant is one which “has a better binding affinityfor human C1q than the parent polypeptide”. Such a molecule may display,for example, about two-fold or more, and preferably about five-fold ormore, improvement in human C1q binding compared to the parentpolypeptide (e.g. at the IC₅₀ values for these two molecules). Forexample, human C1q binding may be about two-fold to about 500-fold, andpreferably from about two-fold or from about five-fold to about1000-fold improved compared to the parent polypeptide.

To assess complement activation, a complement dependent cytotoxicity(CDC) assay may be performed, e.g. as described in Gazzano-Santoro etal., J. Immunol. Methods 202:163 (1996). Briefly, various concentrationsof the polypeptide variant and human complement may be diluted withbuffer. Cells which express the antigen to which the polypeptide variantbinds may be diluted to a density of ˜1×10⁶ cells/ml. Mixtures ofpolypeptide variant, diluted human, complement and cells expressing theantigen may be added to a flat bottom tissue culture 96 well plate andallowed to incubate for 2 hrs at 37° C. and 5% CO₂ to facilitatecomplement mediated cell lysis. 50 μl of alamar blue (AccumedInternational) may then be added to each well and incubated overnight at37° C. The absorbance is measured using a 96-well fluorometer withexcitation at 530 nm and emission at 590 nm. The results may beexpressed in relative fluorescence units (RFU). The sampleconcentrations may be computed from a standard curve and the percentactivity as compared to nonvariant polypeptide is reported for thepolypeptide variant of interest.

Yet another exemplary variant “does not activate complement”. Forexample, 0.6 μg/ml of the polypeptide variant displays about 0-10% CDCactivity in this assay compared to a 0.6 μg/ml of a control antibodyhaving a nonmutated IgG1 Fc region. Preferably the variant does notappear to have any CDC activity in the above CDC assay.

The invention also pertains to a polypeptide variant with enhanced CDCcompared to a parent polypeptide, e.g., displaying about two-fold toabout 100-fold improvement in CDC activity in vitro or in vivo (e.g. atthe IC₅₀ values for each molecule being compared).

A. Receptor Binding Assay and Immune Complex

A receptor binding assay has been developed herein which is particularlyuseful for determining binding of an analyte of interest to a receptorwhere the affinity of the analyte for the receptor is relatively weak,e.g. in the micromolar range as is the case for FcγRIIA, FcγRIIB,FcγRIIIA and FcγRIIIB. The method involves the formation of a molecularcomplex that has an improved avidity for the receptor of interestcompared to the noncomplexed analyte. The preferred molecular complex isan immune complex comprising: (a) an Fc region-containing polypeptide(such as an antibody or an immunoadhesin); (b) a first target moleculewhich comprises at least two binding sites for the Fc region-containingpolypeptide; and (c) a second target molecule which comprises at leasttwo binding sites for the first target molecule.

In Example 1 below, the Fc region-containing polypeptide is an anti-IgEantibody, such as the E27 antibody (FIGS. 4A-4B). E27, when mixed withhuman IgE at an 1:1 molar ratio, forms a stable hexamer consisting ofthree E27 molecules and three IgE molecules. In Example 1 below, the“first target molecule” is a chimeric form of IgE in which the Fabportion of an anti-VEGF antibody is fused to the human IgE Fc portionand the “second target molecule” is the antigen to which the Fab binds(i.e. VEGF). Each molecule of IgE binds two molecules of VEGF. VEGF alsobinds two molecules of IgE per molecule of VEGF. When recombinant humanVEGF was added at a 2:1 molar ratio to IgE:E27 hexamers, the hexamerswere linked into larger molecular weight complexes via the IgE:VEGFinteraction (FIG. 5). The Fc region of the anti-IgE antibody of theresultant immune complex binds to FcR with higher avidity than eitheruncomplexed anti-IgE or anti-IgE:IgE hexamers.

Other forms of molecular complexes for use in the receptor assay arecontemplated. Examples comprising only an Fc region-containingpolypeptide:first target molecule combination include animmunoadhesin:ligand combination such as VEGF receptor(KDR)-immunoadhesin:VEGF and a full-length bispecific antibody(bsAb):first target molecule. A further example of an Fcregion-containing polypeptide:first target molecule:second targetmolecule combination include a nonblocking antibody:solublereceptor:ligand combination such as anti-Trk antibody:soluble Trkreceptor:neurotrophin (Urfer et al. J. Biol. Chem. 273(10):5829-5840(1998)).

Aside from use in a receptor binding assay, the immune complexesdescribed above have further uses including evaluation of Fcregion-containing polypeptide function and immune complex clearance invivo. Hence, the immune complex may be administered to a mammal (e.g. ina pre-clinical animal study) and evaluated for its half-life etc.

To determine receptor binding, a polypeptide comprising at least thebinding domain of the receptor of interest (e.g. the extracellulardomain of an α subunit of an FcR) may be coated on solid phase, such asan assay plate. The binding domain of the receptor alone or areceptor-fusion protein may be coated on the plate using standardprocedures. Examples of receptor-fusion proteins includereceptor-glutathione S-transferase (GST) fusion protein, receptor-chitinbinding domain fusion protein, receptor-hexaHis tag fusion protein(coated on glutathione, chitin, and nickel coated plates, respectively).Alternatively, a capture molecule may be coated on the assay plate andused to bind the receptor-fusion protein via the non-receptor portion ofthe fusion protein. Examples include anti-hexaHis F(ab′)₂ coated on theassay plate used to capture receptor-hexaHis tail fusion or anti-GSTantibody coated on the assay plate used to capture a receptor-GSTfusion. In other embodiments, binding to cells expressing at least thebinding domain of the receptor may be evaluated. The cells may benaturally occurring hematopoietic cells that express the FcR of interestor may be transformed with nucleic acid encoding the FcR or a bindingdomain thereof such that the binding domain is expressed at the surfaceof the cell to be tested.

The immune complex described hereinabove is added to the receptor-coatedplates and incubated for a sufficient period of time such that theanalyte binds to the receptor. Plates may then be washed to removeunbound complexes, and binding of the analyte may be detected accordingto known methods. For example, binding may be detected using a reagent(e.g. an antibody or fragment thereof) which binds specifically to theanalyte, and which is optionally conjugated with a detectable label(detectable labels and methods for conjugating them to polypeptides aredescribed below in the section entitled “Non-Therapeutic Uses for thePolypeptide Variant”).

As a matter of convenience, the reagents can be provided in an assaykit, i.e., a packaged combination of reagents, for combination with theanalyte in assaying the ability of the analyte to bind to a receptor ofinterest. The components of the kit will generally be provided inpredetermined ratios. The kit may provide the first target moleculeand/or the second target molecule, optionally complexed together. Thekit may further include assay plates coated with the receptor or abinding domain thereof (e.g. the extracellular domain of the α subunitof an FcR). Usually, other reagents, such as an antibody that bindsspecifically to the analyte to be assayed, labeled directly orindirectly with an enzymatic label, will also be provided in the kit.Where the detectable label is an enzyme, the kit will include substratesand cofactors required by the enzyme (e.g. a substrate precursor whichprovides the detectable chromophore or fluorophore). In addition, otheradditives may be included such as stabilizers, buffers (e.g. assayand/or wash lysis buffer) and the like. The relative amounts of thevarious reagents may be varied widely to provide for concentrations insolution of the reagents that substantially optimize the sensitivity ofthe assay. Particularly, the reagents may be provided as dry powders,usually lyophilized, including excipients that on dissolution willprovide a reagent solution having the appropriate concentration. The kitalso suitably includes instructions for carrying out the assay.

B. Antibody Preparation

In the preferred embodiment of the invention, the Fc region-containingpolypeptide which is modified according to the teachings herein is anantibody. Techniques for producing antibodies follow:

(i) Antigen Selection and Preparation

Where the polypeptide is an antibody, it is directed against an antigenof interest. Preferably, the antigen is a biologically importantpolypeptide and administration of the antibody to a mammal sufferingfrom a disease or disorder can result in a therapeutic benefit in thatmammal. However, antibodies directed against nonpolypeptide antigens(such as tumor-associated glycolipid antigens; see U.S. Pat. No.5,091,178) are also contemplated.

Where the antigen is a polypeptide, it may be a transmembrane molecule(e.g. receptor) or ligand such as a growth factor. Exemplary antigensinclude molecules such as renin; a growth hormone, including humangrowth hormone and bovine growth hormone; growth hormone releasingfactor; parathyroid hormone; thyroid stimulating hormone; lipoproteins;alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin;follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon;clotting factors such as factor VIIIC, factor IX, tissue factor (TF),and von Willebrands factor; anti-clotting factors such as Protein C;atrial natriuretic factor; lung surfactant; a plasminogen activator,such as urokinase or human urine or tissue-type plasminogen activator(t-PA); bombesin; thrombin; hemopoietic growth factor; tumor necrosisfactor-alpha and -beta; enkephalinase; RANTES (regulated on activationnormally T-cell expressed and secreted); human macrophage inflammatoryprotein (MIP-1-alpha); a serum albumin such as human serum albumin;Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; a microbial protein,such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associatedantigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelialgrowth factor (VEGF); receptors for hormones or growth factors; proteinA or D; rheumatoid factors; a neurotrophic factor such as bone-derivedneurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4,NT-5, or NT-6), or a nerve growth factor such as NGF-β; platelet-derivedgrowth factor (PDGF); fibroblast growth factor such as aFGF and bFGF;epidermal growth factor (EGF); transforming growth factor (TGF) such asTGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, orTGF-β5; insulin-like growth factor-I and -II (IGF-I and IGF-II);des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor bindingproteins; CD proteins such as CD3, CD4, CD8, CD19 and CD20;erythropoietin; osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon such as interferon-alpha,-beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF,GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxidedismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor; viral antigen such as, for example, a portion ofthe AIDS envelope; transport proteins; homing receptors; addressins;regulatory proteins; integrins such as CD11a, CD11b, CD11c, CD18, anICAM, VLA-4 and VCAM; a tumor associated antigen such as HER2, HER3 orHER4 receptor; and fragments of any of the above-listed polypeptides.

Preferred molecular targets for antibodies encompassed by the presentinvention include CD proteins such as CD3, CD4, CD8, CD19, CD20 andCD34; members of the ErbB receptor family such as the EGF receptor,HER2, HER3 or HER4 receptor; cell adhesion molecules such as LFA-1,Mac1, p150.95, VLA-4, ICAM-1, VCAM, α4/β7 integrin, and αv/β3 integrinincluding either α or β subunits thereof (e.g. anti-CD11a, anti-CD18 oranti-CD11b antibodies); growth factors such as VEGF; tissue factor (TF);alpha interferon (α-IFN); an interleukin; such as IL-8; IgE; blood groupantigens; flk2/flt3 receptor; obesity (OB) receptor; mpl receptor;CTLA-4; protein C etc.

Soluble antigens or fragments thereof, optionally conjugated to othermolecules, can be used as immunogens for generating antibodies. Fortransmembrane molecules, such as receptors, fragments of these (e.g. theextracellular domain of a receptor) can be used as the immunogen.Alternatively, cells expressing the transmembrane molecule can be usedas the immunogen. Such cells can be derived from a natural source (e.g.cancer cell lines) or may be cells which have been transformed byrecombinant techniques to express the transmembrane molecule. Otherantigens and forms thereof useful for preparing antibodies will beapparent to those in the art.

(ii) Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen to a protein that is immunogenic in the species to be immunized,e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, whereR and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Preferably, the animal is boostedwith the conjugate of the same antigen, but conjugated to a differentprotein and/or through a different cross-linking reagent. Conjugatesalso can be made in recombinant cell culture as protein fusions. Also,aggregating agents such as alum are suitably used to enhance the immuneresponse.

(iii) Monoclonal Antibodies

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster or macaque monkey, is immunized as hereinabove described toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the monoclonal antibodies). The hybridoma cells serve asa preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transfected into host cells suchas E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells,or myeloma cells that do not otherwise produce immunoglobulin protein,to obtain the synthesis of monoclonal antibodies in the recombinant hostcells. Recombinant production of antibodies will be described in moredetail below.

In a further embodiment, antibodies or antibody fragments can beisolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552-554 (1990). Clackson etal., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.,222:581-597 (1991) describe the isolation of murine and humanantibodies, respectively, using phage libraries. Subsequent publicationsdescribe the production of high affinity (nM range) human antibodies bychain shuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), aswell as combinatorial infection and in vivo recombination as a strategyfor constructing very large phage libraries (Waterhouse et al., Nuc.Acids. Res., 21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, etal., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

(iv) Humanized and Human Antibodies

A humanized antibody has one or more amino acid residues introduced intoit from a source which is non-human. These non-human amino acid residuesare often referred to as “import” residues, which are typically takenfrom an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.,Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody (Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901(1987)). Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

Alternatively, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann etal., Year in Immuno., 7:33 (1993); and Duchosal et al. Nature 355:258(1992). Human antibodies can also be derived from phage-displaylibraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks etal., J. Mol. Biol., 222:581-597 (1991); Vaughan et al. Nature Biotech14:309 (1996)).

(v) Multispecific Antibodies

Multispecific antibodies have binding specificities for at least twodifferent antigens. While such, molecules normally will only bind twoantigens (i.e. bispecific antibodies, BsAbs), antibodies with additionalspecificities such as trispecific antibodies are encompassed by thisexpression when used herein. Examples of BsAbs include those with onearm directed against a tumor cell antigen and the other arm directedagainst a cytotoxic trigger molecule such as anti-FcγRI/anti-CD15,anti-p185^(HER2)/FcγRIII (CD16), anti-CD3/anti-malignant B-cell (1D10),anti-CD3/anti-p185^(HER2), anti-CD3/anti-p97, anti-CD3/anti-renal cellcarcinoma, anti-CD3/anti-OVCAR-3, anti-CD3/L-D1 (anti-colon carcinoma),anti-CD3/anti-melanocyte stimulating hormone analog, anti-EGFreceptor/anti-CD3, anti-CD3/anti-CAMA1, anti-CD3/anti-CD19,anti-CD3/MoV18, anti-neural cell adhesion molecule (NCAM)/anti-CD3,anti-folate binding protein (FBP)/anti-CD3, anti-pan carcinomaassociated antigen (AMOC-31)/anti-CD3; BsAbs with one arm which bindsspecifically to a tumor antigen and one arm which binds to a toxin suchas anti-saporin/anti-Id-1, anti-CD22/anti-saporin,anti-CD7/anti-saporin, anti-CD38/anti-saporin, anti-CEA/anti-ricin Achain, anti-interferon-α (IFN-α)/anti-hybridoma idiotype,anti-CEA/anti-vinca alkaloid; BsAbs for converting enzyme activatedprodrugs such as anti-CD30/anti-alkaline phosphatase (which catalyzesconversion of mitomycin phosphate prodrug to mitomycin alcohol); BsAbswhich can be used as fibrinolytic agents such as anti-fibrin/anti-tissueplasminogen activator (tPA), anti-fibrin/anti-urokinase-type plasminogenactivator (uPA); BsAbs for targeting immune complexes to cell surfacereceptors such as anti-low density lipoprotein (LDL)/anti-Fc receptor(e.g. FcγRI, FcγRII or FcγRIII); BsAbs for use in therapy of infectiousdiseases such as anti-CD3/anti-herpes simplex virus (HSV), anti-T-cellreceptor:CD3 complex/anti-influenza, anti-FcγR/anti-HIV; BsAbs for tumordetection in vitro or in vivo such as anti-CEA/anti-EOTUBE,anti-CEA/anti-DPTA, anti-p185^(HER2)/anti-hapten; BsAbs as vaccineadjuvants; and BsAbs as diagnostic tools such as anti-rabbitIgG/anti-ferritin, anti-horse radish peroxidase (HRP)/anti-hormone,anti-somatostatin/anti-substance P, anti-HRP/anti-FITC,anti-CEA/anti-β-galactosidase. Examples of trispecific antibodiesinclude anti-CD3/anti-CD4/anti-CD37, anti-CD3/anti-CD5/anti-CD37 andanti-CD3/anti-CD8/anti-CD37. Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g. F(ab′)₂ bispecificantibodies).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different-antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is preferred to havethe first heavy-chain constant region (CH1) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986). According to anotherapproach described in W096/27011, the interface between a pair ofantibody molecules can be engineered to maximize the percentage ofheterodimers which are recovered from recombinant cell culture. Thepreferred interface comprises at least a part of the C_(H)3 domain of anantibody constant domain. In this method, one or more small amino acidside chains from the interface of the first antibody molecule arereplaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

While the polypeptide of interest herein is preferably an antibody,other Fc region-containing polypeptides which can be modified accordingto the methods described herein are contemplated. An example of such amolecule is an immunoadhesin.

C. Immunoadhesin Preparation

The simplest and most straightforward immunoadhesin design combines thebinding domain(s) of the adhesin (e.g. the extracellular domain (ECD) ofa receptor) with the Fc region of an immunoglobulin heavy chain.Ordinarily, when preparing the immunoadhesins of the present invention,nucleic acid encoding the binding domain of the adhesin will be fusedC-terminally to nucleic acid encoding the N-terminus of animmunoglobulin constant domain sequence, however N-terminal fusions arealso possible.

Typically, in such fusions the encoded chimeric polypeptide will retainat least functionally active hinge, C_(H)2 and C_(H)3 domains of theconstant region of an immunoglobulin heavy chain. Fusions are also madeto the C-terminus of the Fc portion of a constant domain, or immediatelyN-terminal to the C_(H)1 of the heavy chain or the corresponding regionof the light chain. The precise site at which the fusion is made is notcritical; particular sites are well known and may be selected in orderto optimize the biological activity, secretion, or bindingcharacteristics of the immunoadhesin.

In a preferred embodiment, the adhesin sequence is fused to theN-terminus of the Fc region of immunoglobulin G₁ (IgG₁). It is possibleto fuse the entire heavy chain constant region to the adhesin sequence.However, more preferably, a sequence beginning in the hinge region justupstream of the papain cleavage site which defines IgG Fc chemically(i.e. residue 216, taking the first residue of heavy chain constantregion to be 114), or analogous sites of other immunoglobulins is usedin the fusion. In a particularly preferred embodiment, the adhesin aminoacid sequence is fused to (a) the hinge region and C_(H)2 and C_(H)3 or(b) the C_(H)1, hinge, C_(H)2 and C_(H)3 domains, of an IgG heavy chain.

For bispecific immunoadhesins, the immunoadhesins are assembled asmultimers, and particularly as heterodimers or heterotetramers.Generally, these assembled immunoglobulins will have known unitstructures. A basic four chain structural unit is the form in which IgG,IgD, and IgE exist. A four chain unit is repeated in the highermolecular weight immunoglobulins; IgM generally exists as a pentamer offour basic units held together by disulfide bonds. IgA globulin, andoccasionally IgG globulin, may also exist in multimeric form in serum.In the case of multimer, each of the four units may be the same ordifferent.

Various exemplary assembled immunoadhesins within the scope herein areschematically diagrammed below:

(a) AC_(L)-AC_(L);

(b) AC_(H)-(AC_(H), AC_(L)-AC_(H), AC_(L)-V_(H)C_(H), orV_(L)C_(L)-AC_(H));

(c) AC_(L)-AC_(H)-(AC_(L)-AC_(H), AC_(L)-V_(H)C_(H), V_(L)C_(L)-AC_(H),or V_(L)C_(L)-V_(H)C_(H))

(d) AC_(L)-V_(H)C_(H)-(AC_(H), or AC_(L)-V_(H)C_(H), orV_(L)C_(L)-AC_(H));

(e) V_(L)C_(L)-AC_(H)-(AC_(L)-V_(H)C_(H), or V_(L)C_(L)-AC_(H)); and

(f) (A-Y)_(n)-(V_(L)C_(L)-V_(H)C_(H))₂,

wherein each A represents identical or different adhesin amino acidsequences;

V_(L) is an immunoglobulin light chain variable domain;

V_(H) is an immunoglobulin heavy chain variable domain;

C_(L) is an immunoglobulin light chain constant domain;

C_(H) is an immunoglobulin heavy chain constant domain;

n is an integer greater than 1;

Y designates the residue of a covalent cross-linking agent.

In the interests of brevity, the foregoing structures only show keyfeatures; they do not indicate joining (J) or other domains of theimmunoglobulins, nor are disulfide bonds shown. However, where suchdomains are required for binding activity, they shall be constructed tobe present in the ordinary locations which they occupy in theimmunoglobulin molecules.

Alternatively, the adhesin sequences can be inserted betweenimmunoglobulin heavy chain and light chain sequences, such that animmunoglobulin comprising a chimeric heavy chain is obtained. In thisembodiment, the adhesin sequences are fused to the 3′ end of animmunoglobulin heavy chain in each arm of an immunoglobulin, eitherbetween the hinge and the C_(H)2 domain, or between the C_(H)2 andC_(H)3 domains. Similar constructs have been reported by Hoogenboom, etal., Mol. Immunol. 28:1027-1037 (1991).

Although the presence of an immunoglobulin light chain is not requiredin the immunoadhesins of the present invention, an immunoglobulin lightchain might be present either covalently associated to anadhesin-immunoglobulin heavy chain fusion polypeptide, or directly fusedto the adhesin. In the former case, DNA encoding an immunoglobulin lightchain is typically coexpressed with the DNA encoding theadhesin-immunoglobulin heavy chain fusion protein. Upon secretion, thehybrid heavy chain and the light chain will be covalently associated toprovide an immunoglobulin-like structure comprising two disulfide-linkedimmunoglobulin heavy chain-light chain pairs. Methods suitable for thepreparation of such structures are, for example, disclosed in U.S. Pat.No. 4,816,567, issued 28 Mar. 1989.

Immunoadhesins are most conveniently constructed by fusing the cDNAsequence encoding the adhesin portion in-frame to an immunoglobulin cDNAsequence. However, fusion to genomic immunoglobulin fragments can alsobe used (see, e.g. Aruffo et al., Cell 61:1303-1313 (1990); andStamenkovic et al., Cell 66:1133-1144 (1991)). The latter type of fusionrequires the presence of Ig regulatory sequences for expression. cDNAsencoding IgG heavy-chain constant regions can be isolated based onpublished sequences from cDNA libraries derived from spleen orperipheral blood lymphocytes, by hybridization or by polymerase chainreaction (PCR) techniques. The cDNAs encoding the “adhesin” and theimmunoglobulin parts of the immunoadhesin are inserted in tandem into aplasmid vector that directs efficient expression in the chosen hostcells.

D. Vectors, Host Cells and Recombinant Methods

The invention also provides isolated nucleic acid encoding a polypeptidevariant as disclosed herein, vectors and host cells comprising thenucleic acid, and recombinant techniques for the production of thepolypeptide variant.

For recombinant production of the polypeptide variant, the nucleic acidencoding it is isolated and inserted into a replicable vector forfurther cloning (amplification of the DNA) or for expression. DNAencoding the polypeptide variant is readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the polypeptidevariant). Many vectors are available. The vector components generallyinclude, but are not limited to, one or more of the following: a signalsequence, an origin of replication, one or more marker genes, anenhancer element, a promoter, and a transcription termination sequence.

(i) Signal Sequence Component

The polypeptide variant of this invention may be produced recombinantlynot only directly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The heterologous signal sequence selected preferably isone that is recognized and processed (i.e., cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process the native polypeptide variant signal sequence,the signal sequence is substituted by a prokaryotic signal sequenceselected, for example, from the group of the alkaline phosphatase,penicillinase, Ipp, or heat-stable enterotoxin II leaders. For yeastsecretion the native signal sequence may be substituted by, e.g., theyeast invertase leader, α factor leader (including Saccharomyces andKluyveromyces α-factor leaders), or acid phosphatase leader, the C.albicans glucoamylase leader, or the signal described in WO 90/13646. Inmammalian cell expression, mammalian signal sequences as well as viralsecretory leaders, for example, the herpes simplex gD signal, areavailable.

The DNA for such precursor region is ligated in reading frame to DNAencoding the polypeptide variant.

(ii) Origin of Replication Component

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

(iii) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding β-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up thepolypeptide variant nucleic acid, such as DHFR, thymidine kinase,metallothionein-I and -II, preferably primate metallothionein genes,adenosine deaminase, ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity.

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding polypeptide variant, wild-type DHFR protein, and anotherselectable marker such as aminoglycoside 3′-phosphotransferase (APH) canbe selected by cell growth in medium containing a selection agent forthe selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trp1lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8:135(1990). Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains of Kluyveromyceshave also been disclosed. Fleer et al., Bio/Technology, 9:968-975(1991).

(iv) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to thepolypeptide variant nucleic acid. Promoters suitable for use withprokaryotic hosts include the phoA promoter, β-lactamase and lactosepromoter systems, alkaline phosphatase, a tryptophan (trp) promotersystem, and hybrid promoters such as the tac promoter. However, otherknown bacterial promoters are suitable. Promoters for use in bacterialsystems also will contain a Shine-Dalgarno (S.D.) sequence operablylinked to the DNA encoding the polypeptide variant.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phospho-fructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

Polypeptide variant transcription from vectors in mammalian host cellsis controlled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and most preferablySimian Virus 40 (SV40), from heterologous mammalian promoters, e.g., theactin promoter or an immunoglobulin promoter, from heat-shock promoters,provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the rous sarcoma virus long terminal repeat can be used as the promoter.

(v) Enhancer Element Component

Transcription of a DNA encoding the polypeptide variant of thisinvention by higher eukaryotes is often increased by inserting anenhancer sequence into the vector. Many enhancer sequences are now knownfrom mammalian genes (globin, elastase, albumin, α-fetoprotein, andinsulin). Typically, however, one will use an enhancer from a eukaryoticcell virus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to thepolypeptide variant-encoding sequence, but is preferably located at asite 5′ from the promoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding the polypeptide variant. One usefultranscription termination component is the bovine growth hormonepolyadenylation region. See WO94/11026 and the expression vectordisclosed therein.

(vii) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for polypeptidevariant-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated polypeptidevariant are derived from multicellular organisms. Examples ofinvertebrate cells include plant and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti(mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to thepresent invention, particularly for transfection of Spodopterafrugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can also be utilized as hosts.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/−DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for polypeptide variant production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences.

(viii) Culturing the Host Cells

The host cells used to produce the polypeptide variant of this inventionmay be cultured in a variety of media. Commercially available media suchas Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma),RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM),Sigma) are suitable for culturing the host cells. In addition, any ofthe media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes etal., Anal. Biochem. 102:255 (1980), U.S. Pat. No. 4,767,704; 4,657,866;4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S.Pat. No. Re. 30,985 may be used as culture media for the host cells. Anyof these media may be supplemented as necessary with hormones and/orother growth factors (such as insulin, transferrin, or epidermal growthfactor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleotides (such as adenosine andthymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

(ix) Polypeptide Variant Purification

When using recombinant techniques, the polypeptide variant can beproduced intracellularly, in the periplasmic space, or directly secretedinto the medium. If the polypeptide variant is produced intracellularly,as a first step, the particulate debris, either host cells or lysedfragments, is removed, for example, by centrifugation orultrafiltration. Carter et al., Bio/Technology 10:163-167 (1992)describe a procedure for isolating antibodies which are secreted to theperiplasmic space of E. coli. Briefly, cell paste is thawed in thepresence of sodium acetate (pH 3.5), EDTA, andphenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris canbe removed by centrifugation. Where the polypeptide variant is secretedinto the medium, supernatants from such expression systems are generallyfirst concentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis and antibiotics may be includedto prevent the growth of adventitious contaminants.

The polypeptide variant composition prepared from the cells can bepurified using, for example, hydroxylapatite chromatography, gelelectrophoresis, dialysis, and affinity chromatography, with affinitychromatography being the preferred purification technique. Thesuitability of protein A as an affinity ligand depends on the speciesand isotype of any immunoglobulin Fc region that is present in thepolypeptide variant. Protein A can be used to purify polypeptidevariants that are based on human γ1, γ2, or γ4 heavy chains (Lindmark etal., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for allmouse isotypes and for human γ3 (Guss et al., EMBO J. 5:15671575(1986)). The matrix to which the affinity ligand is attached is mostoften agarose, but other matrices are available. Mechanically stablematrices such as controlled pore glass or poly(styrenedivinyl)benzeneallow for faster flow rates and shorter processing times than can beachieved with agarose. Where the polypeptide variant comprises a C_(H)3domain, the Bakerbond ABX™ resin (J. T. Baker, Phillipsburg, N.J.) isuseful for purification. Other techniques for protein purification suchas fractionation on an ion-exchange column, ethanol precipitation,Reverse Phase HPLC, chromatography on silica, chromatography on heparinSEPHAROSE™ chromatography on an anion or cation exchange resin (such asa polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammoniumsulfate precipitation are also available depending on the polypeptidevariant to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe polypeptide variant of interest and contaminants may be subjected tolow pH hydrophobic interaction chromatography using an elution buffer ata pH between about 2.5-4.5, preferably performed at low saltconcentrations (e.g., from about 0-0.25M salt).

E. Pharmaceutical Formulations

Therapeutic formulations of the polypeptide variant are prepared forstorage by mixing the polypeptide variant having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)), in the form of lyophilized formulations or aqueoussolutions. Acceptable carriers, excipients, or stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptide; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the polypeptide variant, which matricesare in the form of shaped articles, e.g., films, or microcapsule.Examples of sustained-release matrices include polyesters, hydrogels(for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated antibodiesremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for stabilization depending on the mechanisminvolved. For example, if the aggregation mechanism is discovered to beintermolecular S—S bond formation through thio-disulfide interchange,stabilization may be achieved by modifying sulfhydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions.

F. Non-Therapeutic Uses for the Polypeptide Variant

The polypeptide variant of the invention may be used as an affinitypurification agent. In this process, the polypeptide variant isimmobilized on a solid phase such a Sephadex resin or filter paper,using methods well known in the art. The immobilized polypeptide variantis contacted with a sample containing the antigen to be purified, andthereafter the support is washed with a suitable solvent that willremove substantially all the material in the sample except the antigento be purified, which is bound to the immobilized polypeptide variant.Finally, the support is washed with another suitable solvent, such asglycine buffer, pH 5.0, that will release the antigen from thepolypeptide variant.

The polypeptide variant may also be useful in diagnostic assays, e.g.,for detecting expression of an antigen of interest in specific cells,tissues, or serum.

For diagnostic applications, the polypeptide variant typically will belabeled with a detectable moiety. Numerous labels are available whichcan be generally grouped into the following categories:

(a) Radioisotopes, such as ³⁵S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I. The polypeptidevariant can be labeled with the radioisotope using the techniquesdescribed in Current Protocols in Immunology, Volumes 1 and 2, Coligenet al., Ed. Wiley-Interscience, New York, N.Y., Pubs. (1991) for exampleand radioactivity can be measured using scintillation counting.

(b) Fluorescent labels such as rare earth chelates (europium chelates)or fluorescein and its derivatives, rhodamine and its derivatives,dansyl, Lissamine, phycoerythrin and Texas Red are available. Thefluorescent labels can be conjugated to the polypeptide variant usingthe techniques disclosed in Current Protocols in Immunology, supra, forexample. Fluorescence can be quantified using a fluorimeter.

(c) Various enzyme-substrate labels are available and U.S. Pat. No.4,275,149 provides a review of some of these. The enzyme generallycatalyzes a chemical alteration of the chromogenic substrate that can bemeasured using various techniques. For example, the enzyme may catalyzea color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light which can be measured (using achemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g.,firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase,β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et al.,Methods for the Preparation of Enzyme-Antibody Conjugates for use inEnzyme Immunoassay, in Methods in Enzym. (ed J. Langone & H. VanVunakis), Academic press, New York, 73:147-166 (1981).

Examples of enzyme-substrate combinations include, for example:

(i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as asubstrate, wherein the hydrogen peroxidase oxidizes a dye precursor(e.g., orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidinehydrochloride (TMB));

(ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate aschromogenic substrate; and

(iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g.,p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate4-methylumbelliferyl-β-D-galactosidase.

Numerous other enzyme-substrate combinations are available to thoseskilled in the art. For a general review of these, see U.S. Pat. Nos.4,275,149 and 4,318,980.

Sometimes, the label is indirectly conjugated with the polypeptidevariant. The skilled artisan will be aware of various techniques forachieving this. For example, the polypeptide variant can be conjugatedwith biotin and any of the three broad categories of labels mentionedabove can be conjugated with avidin, or vice versa. Biotin bindsselectively to avidin and thus, the label can be conjugated with thepolypeptide variant in this indirect manner. Alternatively, to achieveindirect conjugation of the label with the polypeptide variant, thepolypeptide variant is conjugated with a small hapten (e.g., digoxin)and one of the different types of labels mentioned above is conjugatedwith an anti-hapten polypeptide variant (e.g., anti-digoxin antibody).Thus, indirect conjugation of the label with the polypeptide variant canbe achieved.

In another embodiment of the invention, the polypeptide variant need notbe labeled, and the presence thereof can be detected using a labeledantibody which binds to the polypeptide variant.

The polypeptide variant of the present invention may be employed in anyknown assay method, such as competitive binding assays, direct andindirect sandwich assays, and immunoprecipitation assays. Zola,Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press,Inc. 1987).

The polypeptide variant may also be used for in vivo diagnostic assays.Generally, the polypeptide variant is labeled with a radionuclide (suchas ¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ¹²⁵I, ³H, ³²P or ³⁵S) so that the antigen orcells expressing it can be localized using immunoscintiography.

G. In Vivo Uses for the Polypeptide Variant

It is contemplated that the polypeptide variant of the present inventionmay be used to treat a mammal e.g. a patient suffering from, orpredisposed to, a disease or disorder who could benefit fromadministration of the polypeptide variant. The conditions which can betreated with the polypeptide variant are many and include cancer (e.g.where the polypeptide variant binds the HER2 receptor, CD20 or vascularendothelial growth factor (VEGF)); allergic conditions such as asthma(with an anti-IgE antibody); and LFA-1-mediated disorders (e.g. wherethe polypeptide variant is an anti-LFA-1 or anti-ICAM-1 antibody) etc.

Where the antibody binds the HER2 receptor, the disorder preferably isHER2-expressing cancer, e.g. a benign or malignant tumor characterizedby overexpression of the HER2 receptor. Such cancers include, but arenot limited to, breast cancer, squamous cell cancer, small-cell lungcancer, non-small cell lung cancer, gastrointestinal cancer, pancreaticcancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer,hepatoma, colon cancer, colorectal cancer, endometrial carcinoma,salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer,vulval cancer, thyroid cancer, hepatic carcinoma and various types ofhead and neck cancer. According to the teachings herein, one may preparea polypeptide with a variant Fc region which has improved, ordiminished, ADCC activity. Such molecules will find applications in thetreatment of different disorders.

For example, the polypeptide variant with improved ADCC activity may beemployed in the treatment of diseases or disorders where destruction orelimination of tissue or foreign micro-organisms is desired. Forexample, the polypeptide may be used to treat cancer; inflammatorydisorders; infections (e.g. bacterial, viral, fungal or yeastinfections); and other conditions (such as goiter) where removal oftissue is desired, etc.

Where the polypeptide variant has diminished ADCC activity, suchvariants may be used to treat diseases or disorders where a Fcregion-containing polypeptide with long half-life is desired, but thepolypeptide preferably does not have undesirable effector function(s).For example, the Fc region-containing polypeptide may be an anti-tissuefactor (TF) antibody; anti-IgE antibody; and anti-integrin antibody(e.g. an anti-α4β7 antibody). The desired mechanism of action of such Fcregion-containing polypeptides may be to block ligand-receptor bindingpairs. Moreover, the Fc-region containing polypeptide with diminishedADCC activity may be an agonist antibody.

The polypeptide variant is administered by any suitable means, includingparenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal, and, if desired for local immunosuppressive treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In addition, the polypeptide variant issuitably administered by pulse infusion, particularly with decliningdoses of the polypeptide variant. Preferably the dosing is given byinjections, most preferably intravenous or subcutaneous injections,depending in part on whether the administration is brief or chronic.

For the prevention or treatment of disease, the appropriate dosage ofpolypeptide variant will depend on the type of disease to be treated,the severity and course of the disease, whether the polypeptide variantis administered for preventive or therapeutic purposes, previoustherapy, the patient's clinical history and response to the polypeptidevariant, and the discretion of the attending physician. The polypeptidevariant is suitably administered to the patient at one time or over aseries of treatments.

Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g., 0.1-20 mg/kg) of polypeptide variant is an initialcandidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventional techniquesand assays.

The polypeptide variant composition will be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The“therapeutically effective amount” of the polypeptide variant to beadministered will be governed by such considerations, and is the minimumamount necessary to prevent, ameliorate, or treat a disease or disorder.The polypeptide variant need not be, but is optionally formulated withone or more agents currently used to prevent or treat the disorder inquestion. The effective amount of such other agents depends on theamount of polypeptide variant present in the formulation, the type ofdisorder or treatment, and other factors discussed above. These aregenerally used in the same dosages and with administration routes asused hereinbefore or about from 1 to 99% of the heretofore employeddosages.

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of this invention. All literature and patent citationsmentioned herein are expressly incorporated by reference.

EXAMPLE 1 Low Affinity Receptor Binding Assay

This assay determines binding of an IgG Fc region to recombinantFcγRIIA, FcγRIIB and FcγRIIIA α subunits expressed as His6-glutathione Stransferase (GST)-tagged fusion proteins. Since the affinity of the Fcregion of IgG1 for the FcγRI is in the nanomolar range, the binding ofIgG1 Fc variants can be measured by titrating monomeric IgG andmeasuring bound IgG with a polyclonal anti-IgG in a standard ELISAformat (Example 2 below). The affinity of the other members of the FcγRfamily, i.e. FcγRIIA, FcγRIIB and FcγRIIIA for IgG is however in themicromolar range and binding of monomeric IgG1 for these receptors cannot be reliably measured in an ELISA format.

The following assay utilizes Fc variants of recombinant anti-IgE E27(FIGS. 4A and 4B) which, when mixed with human IgE at a 1:1 molar ratio,forms a stable hexamer consisting of three anti-IgE molecules and threeIgE molecules. A recombinant chimeric form of IgE (chimeric IgE) wasengineered and consists of a human IgE Fc region and the Fab of ananti-VEGF antibody (Presta et al. Cancer Research 57:4593-4599 (1997))which binds two VEGF molecules per mole of anti-VEGF. When recombinanthuman VEGF is added at a 2:1 molar ratio to chimeric IgE:E27 hexamers,the hexamers are linked into larger molecular weight complexes via thechimeric IgE Fab:VEGF interaction. The E27 component of this complexbinds to the FcγRIIA, FcγRIIB and FcγRIIIA α subunits with higheravidity to permit detection in an ELISA format.

Materials and Methods

Receptor Coat:

Fcγreceptor α subunits were expressed as GST fusions of His6 taggedextracellular domains (ECDs) in 293 cells resulting in an ECD-6His-GSTfusion protein (Graham et al. J. Gen. Virol. 36:59-74 (1977) and Gormanet al. DNA Prot. Eng. Tech. 2:3-10 (1990)) and purified by Ni-NTA columnchromatography (Qiagen, Australia) and buffer exchanged into phosphatebuffered saline (PBS). Concentrations were determined by absorption at280 nm using extinction coefficients derived by amino acid compositionanalysis. Receptors were coated onto Nunc F96 maxisorb plates (cat no:439454) at 100 ng per well by adding 100 μl of receptor-GST fusion at 1μg/ml in PBS and incubated for 48 hours at 4° C. Prior to assay, platesare washed 3× with 250 μl of wash buffer (PBS pH 7.4 containing 0.5%TWEEN 20™) and blocked with 250 μl of assay buffer (50 mM Tris bufferedsaline, 0.05% TWEEN 20™, 0.5% RIA grade bovine albumin (Sigma A7888),and 2 mM EDTA pH 7.4).

Immune Complex Formation:

Equal molar amounts (1:1) of E27 and recombinant chimeric IgE whichbinds two moles recombinant human VEGF per mole of chimeric IgE areadded to a 12×75 mm polypropylene tube in PBS and mixed by rotation for30 minutes at 25° C. E27 (anti-IgE)/chimeric IgE (IgE) hexamers areformed during this incubation. Recombinant human VEGF (165 form, MW44,000) is added at a 2:1 molar ratio to the IgE concentration and mixedby rotation an additional 30 minutes at 25° C. VEGF-chimeric IgE bindinglinks E27:chimeric IgE hexamers into larger molecular weight complexeswhich bind FcγR α subunit ECD coated plates via the Fc region of the E27antibody.

E27:Chimeric IgE:VEGF:

(1:1:2 molar ratio) complexes are added to FcγR α subunit coated platesat E27 concentrations of 5 μg and 1 μg total IgG in quadruplicate inassay buffer and incubated for 120 minutes at 25° C. on an orbitalshaker.

Complex Detection:

Plates are washed 5× with wash buffer to remove unbound complexes andIgG binding is detected by adding 100 μl horse radish peroxidase (HRP)conjugated goat anti-human IgG (γ) heavy chain specific (BoehringerMannheim 1814249) at 1:10,000 in assay buffer and incubated for 90 minat 25° C. on an orbital shaker. Plates are washed 5× with wash buffer toremove unbound HRP goat anti-human IgG and bound anti-IgG is detected byadding 100 μl of substrate solution (0.4 mg/ml o-phenylenediaminedihydrochloride, Sigma P6912, 6 mM H₂O₂ in PBS) and incubating for 8 minat 25° C. Enzymatic reaction is stopped by the addition of 100 μl 4.5NH₂SO₄ and colorimetric product is measured at 490 nm on a 96 well platedensitometer (Molecular Devices). Binding of E27 variant complexes isexpressed as a percent of the wild type E27 containing complex.

EXAMPLE 2 Identification of Unique C1q Binding Sites in a Human IgGAntibody

In the present study, mutations were identified in the CH2 domain of ahuman IgG1 antibody, “C2B8” (Reff et al., Blood 83:435 (1994)), thatablated binding of the antibody to C1q but did not alter theconformation of the antibody nor affect binding to each of the FcγRs. Byalanine scanning mutagenesis, five variants in human IgG1 wereidentified, D270K, D270V, K322A P329A, and P331, that were non-lytic andhad decreased binding to C1q. The data suggested that the core C1qbinding sites in human IgG1 is different from that of murine IgG2b. Inaddition, K322A, P329A and P331A were found to bind normally to the CD20antigen, and to four Fc receptors, FcγRI, FcγRII, FcγRIII and FcRn.

Materials and Methods

Construction of C2B8 Variants:

The chimeric light and heavy chains of anti-CD20-antibody C2B8 (Reff etal., Blood 83:435 (1994)) subcloned separately into previously describedPRK vectors (Gorman et al., DNA Protein Eng. Tech. 2:3 (1990)) wereused. By site directed mutagenesis (Kunkel et al., Proc. Natl. Acad.Sci. USA 82:488 (1987)), alanine scan variants of the Fc region in theheavy chain were constructed. The heavy and light chain plasmids wereco-transfected into an adenovirus transformed human embryonic kidneycell line as previously described (Werther et al., J. Immunol. 157:4986(1996)). The media was changed to serum-free 24 hours after transfectionand the secreted antibody was harvested after five days. The antibodieswere purified using Protein A-SEPHAROSE CL4B™ (Pharmacia), bufferexchanged and concentrated to 0.5 ml with PBS using a Centricon-30(Amicon), and stored at 4° C. The concentration of the antibody wasdetermined using total Ig-binding ELISA.

C1q Binding ELISA:

Costar 96 well plates were coated overnight at 4° C. with the indicatedconcentrations of C2B8 in coating buffer (0.05 M sodium carbonatebuffer), pH 9. The plates were then washed 3× with PBS/0.05% TWEEN 20™,pH 7.4 and blocked with 200 μl of ELISA diluent without thimerosal (0.1MNaPO4/0.1M NaCl/0.1% gelatin/0.05% TWEEN 20™/0.05% ProClin300) for 1 hrat room temperature. The plate was washed 3× with wash buffer, analiquot of 100 μl of 2 μg/ml C1q (Quidel, San Diego, Calif.) was addedto each well and incubated for 2 hrs at room temperature. The plate wasthen washed 6× with wash buffer. 100 μl of a 1:1000 dilution of sheepanti-complement C1q peroxidase conjugated antibody (Biodesign) was addedto each well and incubated for 1 hour at room temperature. The plate wasagain washed 6× with wash buffer and 100 μl of substrate buffer(PBS/0.012% H₂0₂) containing OPD (O-phenylenediamine dihydrochloride(Sigma)) was added to each well. The oxidation reaction, observed by theappearance of a yellow color, was allowed to proceed for 30 minutes andstopped by the addition of 100 μl of 4.5 N H₂SO₄. The absorbance wasthen read at (492-405) nm using a microplate reader (SPECTRA MAX 250™,Molecular Devices Corp.). The appropriate controls were run in parallel(i.e. the ELISA was performed without C1q for each concentration of C2B8used and also the ELISA was performed without C2B8). For each variant,C1q binding was measured by plotting the absorbance (492-405) nm versusconcentration of C2B8 in μg/ml using a 4-parameter curve fitting program(KALEIDAGRAPH™) and comparing EC₅₀ values.

Complement Dependent Cytotoxicity (CDC) Assay:

This assay was performed essentially as previously described(Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996)). Variousconcentrations of C2B8 (0.08-20 μg/ml) were diluted with RHB buffer(RPMI 1640/20 mM HEPES (pH 7.2)/2 mM Glutamine/0.1% BSA/100 μg/mlGentamicin). Human complement (Quidel) was diluted 1:3 in RHB buffer andWIL2-S cells (available from the ATCC, Manassas, Va.) which express theCD20 antigen were diluted to a density of 1×10⁶ cells/ml with RHBbuffer. Mixtures of 150 μl containing equal volumes of C2B8, dilutedhuman complement and WIL2-S cells were added to a flat bottom tissueculture 96 well plate and allowed to incubate for 2 hrs at 37° C. and 5%CO₂ to facilitate complement mediated cell lysis. 50 μl of alamar blue(Accumed International) was then added to each well and incubatedovernight at 37° C. The absorbance was measured using a 96-wellfluorometer with excitation at 530 nm and emission at 590 nm. Asdescribed by Gazzano-Santoro et al., the results are expressed inrelative fluorescence units (RFU). The sample concentrations werecomputed from a C2B8 standard curve and the percent activity as comparedto wild type C2B8 is reported for each variant.

CD20 Binding Potency of the C2B8 Variants:

The binding of C2B8 and variants to the CD20 antigen were assessed by amethod previously described (Reff et al., (1994), supra; reviewed inGazzano-Santoro et al., (1996), supra). WIL2-S cells were grown for 3-4days to a cell density of 1×10⁶ cells/ml. The cells were washed and spuntwice in FACS buffer (PBS/0.1% BSA/0.02% NaN₃) and resuspended to a celldensity of 5×10⁶ cells/ml. 200 μl of cells (5×10⁶ cells/ml) and 20 μl ofdiluted C2B8 samples were added to a 5 ml tube and incubated at roomtemperature for 30 minutes with agitation. The mixture was then washedwith 2 ml of cold FACS buffer, spun down and resuspended in 200 μl ofcold FACS buffer. To the suspension, 10 μl of goat anti-human IgG-FITC(American Qualex Labs.) was added and the mixture was incubated in thedark at room temperature for 30 minutes with agitation. Afterincubation, the mixture was washed with 2 ml of FACS buffer, spun downand resuspended in 1 ml of cold fixative buffer (1% formaldehyde inPBS). The samples were analyzed by flow cytometry and the resultsexpressed as relative fluorescence units (RFU) were plotted againstantibody concentrations using a 4-parameter curve fitting program(KALEIDAGRAPH™). The EC₅₀ values are reported as a percentage of that ofthe C2B8 reference material.

FcγR Binding ELISAs:

FcγRI α subunit-GST fusion was coated onto Nunc F96 maxisorb plates (catno. 439454) by adding 100 μl of receptor-GST fusion at 1 μg/ml in PBSand incubated for 48 hours at 4° C. Prior to assay, plates are washed 3×with 250 μl of wash buffer (PBS pH 7.4 containing 0.5% TWEEN 20™) andblocked with 250 μl of assay buffer (50 mM Tris buffered saline, 0.05%TWEEN 20™, 0.5% RIA grade bovine albumin (Sigma A7888), and 2 mM EDTA pH7.4). Samples diluted to 10 μg/ml in 1 ml of assay buffer are added toFcγRI α subunit coated plates and incubated for 120 minutes at 25° C. onan orbital shaker. Plates are washed 5× with wash buffer to removeunbound complexes and IgG binding is detected by adding 100 μl horseradish peroxidase (HRP) conjugated goat anti-human IgG (γ) heavy chainspecific (Boehringer Mannheim 1814249) at 1:10,000 in assay buffer andincubated for 90 min at 25° C. on an orbital shaker. Plates are washed5× with wash buffer to remove unbound HRP goat anti-human IgG and boundanti-IgG Is detected by adding 100 μl of substrate solution (0.4 mg/mlo-phenylenediamine dihydrochloride, Sigma P6912, 6 mM H₂O₂ in PBS) andincubating for 8 min at 25° C. Enzymatic reaction is stopped by theaddition of 100 μl 4.5N H₂SO₄ and colorimetric product is measured at490 nm on a 96 well plate densitometer (Molecular Devices). Binding ofvariant is expressed as a percent of the wild type molecule.

FcγRII and III binding ELISAs were performed as described in Example 1above.

For measuring FcRn binding activity of IgG variants, ELISA plates werecoated with 2 μg/ml streptavidin (Zymed, South San Francisco) in 50 mMcarbonate buffer, pH 9.6, at 4° C. overnight and blocked with PBS-0.5%BSA, pH 7.2 at room temperature for one hour. Biotinylated FcRn(prepared using biotin-X-NHS from Research Organics, Cleveland, Ohio andused at 1-2 μg/ml) in PBS-0.5% BSA, 0.05% polysorbate 20, pH 7.2, wasadded to the plate and incubated for one hour. Two fold serial dilutionsof IgG standard (1.6-100 ng/ml) or variants in PBS-0.5% BSA, 0.05%polysorbate 20, pH 6.0, were added to the plate and incubated for twohours. Bound IgG was detected using peroxidase labeled goat F(ab′)₂anti-human IgG F(ab′)₂ in the above pH 6.0 buffer (JacksonImmunoResearch, West Grove, Pa.) followed by 3,3′,5,5′-tetramethylbenzidine (Kirgaard & Perry Laboratories) as the substrate. Plates werewashed between steps with PBS-0.05% polysorbate 20 at either pH 7.2 or6.0. Absorbance was read at 450 nm on a Vmax plate reader (MolecularDevices, Menlo Park, Calif.). Titration curves were fit with afour-parameter nonlinear regression curve-fitting program (KaleidaGraph,Synergy software, Reading, Pa.). Concentrations of IgG variantscorresponding to the mid-point absorbance of the titration curve of thestandard were calculated and then divided by the concentration of thestandard corresponding to the mid-point absorbance of the standardtitration curve.

Results and Discussion

By alanine scanning mutagenesis, several single point mutations wereconstructed in the CH2 domain of C2B8 beginning with E318A, K320A andK322A. All the variants constructed bound normally to the CD20 antigen(Table 3).

TABLE 3 wt E318A K320A K322A P329A P331A FcRn + + + + CD20 + + + + + +FcγRI + + + + + + FcγRII + + + + + + FcγRIII + + + + + + *C1q +++ ++ +++− − − CDC + + + − − − (+) indicates binding and (−) signifies bindingabolished *With respect to C1q binding, each + sign is equivalent toapproximately 33% binding.

Where binding of human complement to an antibody with a human Fc wasanalyzed, the ability of E318A and K320A to activate complement wasessentially identical to that of wild type C2B8 (Table 3). When comparedto wild type C2B8, there appears to be little difference in the bindingof E318A and K320A to C1q. There is only a 10% decrease in the bindingof K320A and about a 30% decrease in the binding of E318A to C1q (FIG.2). The results indicate that the effect of the E318A and the K320Asubstitution on complement activation and C1q binding is minimal. Also,the human IgG1 of C2B8 was substituted for human IgG2 and used as anegative control in the C1q binding studies. The IgG2 variant appears tohave a much lower affinity for C1q than the E318A and K320A variants(FIG. 2). Thus, the results demonstrate that E318 and K320 do notconstitute the core C1q binding sites for human IgG1. Conversely, theK322A substitution had a significant effect on both complement activityand C1q binding. The K322A variant did not have CDC activity when testedin the above CDC assay and was more than a 100 fold lower than wild typeC2B8 in binding to C1q (FIG. 2). In the human system, K322 is the onlyresidue of the proposed core C1q binding sites that appeared to have asignificant effect on complement activation and C1q binding.

Since the Duncan and Winter study was performed using mouse IgG2b andthe above results reveal that K320 and E318 in human IgG1 are notinvolved in C1q binding, and without being bound to any one theory, theabove data suggest that the C1q binding region in murine IgGs isdifferent from that of the human. To investigate this further and alsoto identify additional variants that do not bind to C1q and hence do notactivate complement, several more point mutations in the vicinity ofK322 were constructed as assessed from the three dimensional structureof the C2B8 Fc. Variants constructed, K274A, N276A, Y278A, S324A, P329A,P331A, K334A, and T335A, were assessed for their ability to bind C1q andalso to activate complement. Many of these substitutions had little orno effect on C1q binding or complement activation. In the above assays,the P329A and the P331A variants did not activate complement and haddecreased binding to C1q. The P331A variant did not activate complementand was 60 fold lower in binding to C1q (FIG. 3) when compared to wildtype C2B8 (FIG. 2). The concentration range of the antibody variantsused in FIG. 3 is expanded to 100 μg/ml in order to observe saturationof C1q binding to the P331A variant. The mutation P329A results in anantibody that does not activate complement and is more than a 100 foldlower in binding to C1q (FIG. 3) when compared to wild type C2B8 (FIG.2).

Variants that did not bind to C1q and hence did not activate complementwere examined for their ability to bind to the Fc receptors: FcγRI,FcγRIIA, FcγRIIB, FcγRIIIA and FcRn. This particular study was performedusing a humanized anti-IgE antibody, an IgG1 antibody with thesemutations (see Example 1 above). The results revealed the variants,K322A and P329A, bind to all the Fc receptors to the same extent as thewild type protein (Table 4). However, there was a slight decrease in thebinding of P331A to FcγRIIB.

In conclusion, two amino acid substitutions in the COOH terminal regionof the CH2 domain of human IgG1, K322A and P329A were identified thatresult in more than 100 fold decrease in C1q binding and do not activatethe CDC pathway. These two variants, K322A and P329A, bind to all Fcreceptors with the same affinity as the wild type antibody. Based on theresults, summarized in Table 4, and without being bound to any onetheory, it is proposed that the C1q binding epicenter of human IgG1 iscentered around K322, P329 and P331 and is different from the murineIgG2b epicenter which constitutes E318, K320 and K322.

TABLE 4 wt E318A K320A K322A P329A P331A CD20 100 89 102 86 112 103^(a)FcγRI 100 93 102 90 104 74 ^(a)FcγRIIA 100 113 94 109 111 86^(a)FcγRIIB 100 106 83 101 96 58 ^(a)FcγRIII 100 104 72 90 85 73 CDC 100108 108 none none none ^(a)For binding to the FcγRs the variants weremade in the E27 background (anti-IgE). The results are presented as apercentage of the wild type.

A further residue involved in binding human C1q was identified using themethods described in the present example. The residue D270 was replacedwith lysine and valine to generate variants D270K and D270V,respectively. These variants both showed decreased binding to human C1q(FIG. 6) and were non-lytic (FIG. 7). The two variants bound the CD20antigen normally and recruited ADCC.

EXAMPLE 3 Variants with Improved C1q Binding

The following study shows that substitution of residues at positionsK326, A327, E333 and K334 resulted in variants with at least about a 30%increase in binding to C1q when compared to the wild type antibody. Thisindicated K326, A327, E333 and K334 are potential sites for improvingthe efficacy of antibodies by way of the CDC pathway. The aim of thisstudy was to improve CDC activity of an antibody by increasing bindingto C1q. By site directed mutagenesis at K326 and E333, several variantswith increased binding to C1q were constructed. The residues in order ofincreased binding at K326 are K<V<E<A<G<D<M<W, and the residues in orderof increased binding at E333 are E<Q<D<V<G<A<S. Four variants, K326M,K326D, K326E and E333S were constructed with at least a two-foldincrease in binding to C1q when compared to wild type. Variant K326Wdisplayed about a five-fold increase in binding to C1q.

Variants of the wild type C2B8 antibody were prepared as described abovein Example 2. A further control antibody, wild type C2B8 produced inChinese hamster ovary (CHO) cells essentially as described in U.S. Pat.No. 5,736,137, was included in a C1q binding ELISA to confirm that wtC2B8 produced in the 293 kidney cell line had the same C1q bindingactivity as the CHO-produced antibody (see “CHO-wt-C2B8” in FIG. 8). TheC1q binding ELISA, CDC assay, and CD20 binding potency assay in thisexample were performed as described in Example 2 above.

As shown in FIG. 8, alanine substitution at K326 and E333 in C2B8resulted in variants with about a 30% increase in binding to C1q.

Several other single point variants at K326 and E333 were constructedand assessed for their ability to bind C1q and activate complement. Allthe variants constructed bound normally to the CD20 antigen.

With respect to K326, the other single point variants constructed wereK326A, K326D, K326E, K326G, K326V, K326M and K326W. As shown in FIG. 9,these variants all bound to C1q with a better affinity than the wildtype antibody. K326W, K326M, K326D and K326E showed at least a two-foldincrease in binding to C1q (Table 5). Among the K326 variants, K326W hadthe best affinity for C1q.

TABLE 5 Variant EC₅₀ value Wild type 1.53 K326V 1.30 K326A 1.03 K326E1.08 K326G 0.95 K326D 0.76 K326M 0.67 K326W 0.47 E333S 0.81 E333A 0.98E333G 1.14 E333V 1.18 E333D 1.22 E333Q 1.52 K334A 1.07

Substitutions with hydrophobic as well as charged residues resulted invariants with increased binding to C1q. Even substitution with glycinewhich is known to impart flexibility to a chain and is well conserved innature, resulted in a variant with higher affinity for C1q when comparedto the wild type. It would appear that any amino acid substitution atthis site would result in a variant with higher affinity for C1q. Asassessed from the three-dimensional structure, K326 and E333 are in thevicinity of the core C1q binding sites (FIG. 10).

In addition to alanine, E333 was also substituted with other amino acidresidues. These variants, E333S, E333G, E333V, E333D, and E333Q, all hadincreased binding to C1q when compared to the wild type (FIG. 11). Asshown in Table 5, the order of binding affinity for C1q was as follows:E333S>E333A>E333G>E333V>E333D>E333Q. Substitutions with amino acidresidues with small side chain volumes, i.e. serine, alanine andglycine, resulted in variants with higher affinity for C1q in comparisonto the other variants, E333V, E333D and E333Q, with larger side chainvolumes. The variant E333S had the highest affinity for C1q, showing atwo-fold increase in binding when compared to the wild type. Withoutbeing bound to any one theory, this indicates the effect on C1q bindingat 333 may also be due in part to the polarity of the residue.

Double variants were also generated. As shown in FIGS. 12 and 13, doublevariants K326M-E333S and K326A-E333A were at least three-fold better atbinding human C1q than wild type C2B8 (FIG. 12) and at least two-foldbetter at mediating CDC compared to wild type C2B8 (FIG. 13). Additivityindicates these are independently acting variants.

As shown in FIG. 14, a further variant with improved C1q binding (50%increase) was made by changing A327 in a human IgG1 constant region toglycine. Conversely, in a human IgG2 constant region, changing G327 toalanine reduced C1q binding of the IgG2 antibody.

EXAMPLE 4 Identification of FcR Binding Sites in Human IgG Antibodies

In the present study, the effect of mutating various Fc region residuesof an IgG1 antibody with respect to binding FcγRI, FcγRIIA, FcγRIIB andFcγRIIIIA as well as FcRn was evaluated. Antibody variants with improvedas well as diminished FcR binding were identified.

Materials and Methods

Construction of IgG1 Variants:

Recombinant anti-IgE E27 having the light chain and heavy chainsequences in FIGS. 4A and 4B, respectively, was used as the parentantibody in the following experiments. This antibody binds the antigenIgE and has a non-A allotype IgG1 Fc region. By site directedmutagenesis (Kunkel et al., Proc. Natl. Acad. Sci. USA 82:488 (1987)),variants of the Fc region in the heavy chain of the above parentantibody were constructed. The heavy and light chain plasmids wereco-transfected into an adenovirus transformed human embryonic kidneycell line as previously described (Werther et al., J. Immunol. 157:4986(1996)). The media was changed to serum-free 24 hours after transfectionand the secreted antibody was harvested after five days. The antibodieswere purified by Protein G SEPHAROSE® (Pharmacia), buffer exchanged andconcentrated to 0.5 ml with PBS using a Centricon-30 (Amicon), andstored at 4° C. Concentration was determined by adsorption at 280 nmusing extinction coefficients derived by amino acid compositionanalysis.

High Affinity FcγRIA Binding ELISA:

FcγRIA was expressed as a GST fusion of His6 tagged extracellular domainin 293 cells and purified by Ni-NTA column chromatography.

To purify FcγRIA, supernatant from transfected 293 cells was removedafter three days. Protease inhibitors were added; 50 μL Aprotinin(Sigma)/50 mL supernatant, and PMSF (1 mM). Supernatants wereconcentrated to 10 mL in a stirred cell (Amicon), and dialyzed overnightat 4° C. against 1 liter column buffer (50 mM Tris pH 8.0, 20 mMImidazole, 300 mM NaCl). Additional dialysis was done the followingmorning against fresh column buffer for 4 hours at 4° C. The solutionwas loaded on to a 1 mL Ni⁺⁺ column (NTA super flow resin, Qiagen)previously equilibrated with 10 mL column buffer. Columns were washedwith 10 mL column buffer, and protein was eluted with 2.5 mL elutionbuffer (50 mM Tris pH 8.0, 250 mM Imidazole, 300 mM NaCl). Protein wasconcentrated to 0.5 mL and buffer exchanged into PBS. Concentrationswere determined by adsorption at 280 nm using an extinction coefficientderived by amino acid composition analysis.

Purified receptors were coated onto Nunc F96 maxisorb plates (cat no.439545) at approximately 150 ng per well by adding 100 μL of receptor at1.5 μg/mL in PBS and incubated for 24 hours at 4° C. Prior to assay,plates were washed 3× with 250 μL of wash buffer (phosphate bufferedsaline pH 7.4 containing 0.5% TWEEN 20®) and blocked with 250 μL ofassay buffer (50 mM tris buffered saline, 0.05% TWEEN 20®, 0.5% RIAgrade bovine albumin (Sigma A7888), and 2 mM EDTA pH 7.4).

100 μL of E27 was added to the first four wells of the FcγRIA subunitcoated plated at a concentration of 10 μg/mL. 80 μL of assay buffer wasadded to the next four well followed by 20 μL of the 10 μg/mL E27 IgG togive a final concentration of 2 μg/mL. Plates were incubated at 25° C.for 2 hours on an orbital shaker.

For detection, plates were washed 5× with wash buffer to remove unboundantibody. IgG binding to GST-FcγRIA was detected by adding 100 μL horseradish peroxidase (HRP) conjugated protein G (BIORAD) at 1:5000. HRPconjugates were incubated for 1.5 hours at 25° C. on an orbital shaker.Plates were washed ×5 with wash buffer to remove unbound HRP conjugate.Binding was detected by adding 100 μL of substrate solution (0.4 mg/mLo-phenylenediamine dihydrochloride, Sigma P6912, 6 mM H₂O₂ in PBS) andincubating for 10 minutes at 25° C. Enzymatic reaction was stopped bythe addition of 100 μL of 4.5 N H₂SO₄ and colorimetric product wasmeasured at 490 nm on a 96 well plate densitometer (Molecular Devices).

Binding of E27 variants at IgG concentration of 2 μg/mL was expressed asa ratio of wild type E27.

FcγRIA THP-1 Assay:

100 μL of E27 was added to the first three wells of a serocluster plate(Costar) at a concentration of 20 μg/mL in assay buffer (1×PBS, 0.1%BSA, 0.01% NaN₃). 92.5 μL of assay buffer was added to the next threewells followed by 7.5 μL of the 20 μg/mL E27 IgG to give a finalconcentration of 1.5 μg/mL. To each well, 100 μL of THP-1 cells wereadded at a concentration of 5 million cells/mL in FACS assay buffer. Theplate is incubated on ice for 30 minutes

For detection, cells were washed 2× with assay buffer to remove unboundantibody. IgG binding FcγRIA was detected by adding 100 μL FITCconjugated F(ab′)₂ fragment of goat anti-human IgG heavy chain specific.(Jackson Immunoresearch) at 1:200. FITC conjugates were incubated withcells for 30 minutes on ice. Cells were washed ×3 with assay buffer toremove unbound FITC conjugate. Cells were stained with P.I. (SIGMA) at2.5 μg/mL and analyzed by flow cytometry.

Binding of E27 variants at IgG concentration of 1.5 μg/mL was expressedas a ratio of wild type E27.

Data from the plate assay (FcγRIA ELISA) and cell-based assay (FcγRIATHP-1 assay) was averaged to arrive at an FcγRIA-binding activity.

Low Affinity FcγR Binding ELISAs:

FcγRIIA, FcγRIIB and FcγRIIIA binding ELISAs were performed as describedin Example 1 above, with detection of the stable hexamer (consisting ofthree anti-IgE molecules and three IgE molecules).

Binding Values:

For all FcγR, binding values reported in Table 6 are the binding of eachE27 variant relative to native E27, taken as(A_(490 nm variant)/A_(490 nm native IgG1)) at 0.33 or 1 μg/ml forFcγRII and FcγRIII A and 2 μg/ml for FcγRI. A value greater than 1denotes binding of the variant was improved compared to native IgG1while a ratio less than 1 denotes reduced binding compared to nativeIgG1. Reduced binding to any given receptor was defined as a reductionof ≧40% compared to native IgG; better binding was defined as animprovement of ≧25% compared to native IgG 1. The latter was chosenbased on the observation that variants with ≧25% improved binding in theELISA-format assay, such as Glu333Ala, Lys334Ala and Ser298Ala, alsoshowed improved efficacy in the cell-based binding and ADCC assays.

FcRn Binding ELISA:

For measuring FcRn binding activity of IgG variants, ELISA plates werecoated with 2 μg/ml streptavidin (Zymed, South San Francisco) in 50 mMcarbonate buffer, pH 9.6, at 4° C. overnight and blocked with PBS-0.5%BSA, pH 7.2 at room temperature for one hour. Biotinylated FcRn(prepared using biotin-X—NHS from Research Organics, Cleveland, Ohio andused at 1-2 μg/ml) in PBS-0.5% BSA, 0.05% polysorbate 20, pH 7.2, wasadded to the plate and incubated for one hour. Two fold serial dilutionsof IgG standard (1.6-100 ng/ml) or variants in PBS-0.5% BSA, 0.05%polysorbate 20, pH 6.0, were added to the plate and Incubated for twohours. Bound IgG was detected using peroxidase labeled goat F(ab′)₂anti-human IgG F(ab′)₂ in the above pH 6.0 buffer (JacksonImmunoResearch, West Grove, Pa.) followed by 3,3′,5,5′-tetramethylbenzidine (Kirgaard & Perry Laboratories) as the substrate. Plates werewashed between steps with PBS-0.05% TWEEN 20® at either pH 7.2 or 6.0.Absorbance was read at 450 nm on a Vmax plate reader (Molecular Devices,Menlo Park, Calif.). Titration curves were fit with a four-parameternonlinear regression curve-fitting program (KaleidaGraph, Synergysoftware, Reading, Pa.). Concentrations of IgG variants corresponding tothe mid-point absorbance of the titration curve of the standard werecalculated and then divided by the concentration of the standardcorresponding to the mid-point absorbance of the standard titrationcurve.

In Vitro ADCC Assay:

To prepare chromium 51-labeled target cells, tumor cell lines were grownin tissue culture plates and harvested using sterile 10 mM EDTA in PBS.SK-BR-3 cells, a 3+ HER2-overexpressing human breast cancer cell line,were used as targets in all assays. The detached cells were washed twicewith cell culture medium. Cells (5×10⁶) were labeled with 200 μCi ofchromium51 (New England Nuclear/DuPont) at 37° C. for one hour withoccasional mixing. Labeled cells were washed three times with cellculture medium, then were resuspended to a concentration of 1×10⁵cells/mL. Cells were used either without opsonization, or were opsonizedprior to the assay by incubation with rhuMAb HER2 wildtype (HERCEPTIN®)or seven Fc mutants (G14 D265A; G18 D270A; G17 E269A; G36 S298A; G30K290A; G31 R292A; and G34 Q295A) at 100 ng/mL and 1.25 ng/mL in PBMCassay or 20 ng/mL and 1 ng/mL in NK assay.

Peripheral blood mononuclear cells (PBMCs) were prepared by collectingblood on heparin from normal healthy donors and dilution with an equalvolume of phosphate buffered saline (PBS). The blood was then layeredover LYMPHOCYTE SEPARATION MEDIUM® (LSM: Organon Teknika) andcentrifuged according to the manufacturer's instructions. Mononuclearcells were collected from the LSM-plasma interface and were washed threetimes with PBS. Effector cells were suspended in cell culture medium toa final concentration of 1×10⁷ cells/mL.

After purification through LSM, natural killer (NK) cells were isolatedfrom PBMCs by negative selection using an NK cell isolation kit and amagnetic column (Miltenyi Biotech) according to the manufacturer'sinstructions. Isolated NK cells were collected, washed and resuspendedin cell culture medium to a concentration of 2×10⁶ cells/mL. Theidentity of the NK cells was confirmed by flow cytometric analysis.

Varying effector:target ratios were prepared by serially diluting theeffector (either PBMC or NK) cells two-fold along the rows of amicrotiter plate (100 μL final volume) in cell culture medium. Theconcentration of effector cells ranged from 1.0×10⁷/mL to 2.0×10⁴/mL forPBMC and from 2.0×10⁶/mL to 3.9×10³/mL for NK. After titration ofeffector cells, 100 μL of chromium 51-labeled target cells (opsonized ornonoponsonized) at 1×10⁵ cells/mL were added to each well of the plate.This resulted in an initial effector:target ratio of 100:1 for PBMC and20:1 for NK cells. All assays were run in duplicate, and each platecontained controls for both spontaneous lysis (no effector cells) andtotal lysis (target cells plus 100 μL) 1% sodium dodecyl sulfate, 1 Nsodium hydroxide). The plates were incubated at 37° C. for 18 hours,after which the cell culture supernatants were harvested using asupernatant collection system (Skatron Instrument, Inc.) and counted ina Minaxi auto-gamma 5000 series gamma counter (Packard) for one minute.Results were then expressed as percent cytotoxicity using the formula: %Cytotoxicity=(sample cpm-spontaneous lysis)/(total lysis-spontaneouslysis)×100. Four-parameter curve-fitting was then used to evaluate thedata (KaleidaGraph 3.0.5).

RESULTS

A variety of antibody variants were generated which had FcR bindingactivity that differed from the parent antibody. The FcR binding datafor the variants generated is shown in Tables 6 and 7 below. Anadditional variant, T307Q, also displayed improved FcRn binding comparedto E27 parent antibody.

TABLE 6 Binding of Human IgG1 Variants to Human FcRn and FcγR FcRn^(b)FcγRI FcγRIIA FcγRIIB FcγRIIIA Variant^(a) mean (sd) N mean (sd) N mean(sd) mean (sd) mean sd N^(c) Class 1 = Reduced binding to all FcγRGlu233Pro 0.54 (0.20) 3 0.12 (0.06) 6 0.08 (0.01) 0.12 (0.01) 0.04(0.02) 2 Leu234Val Leu235Ala Gly236deleted Pro238Ala 1.49 (0.17) 3 0.60(0.05) 5 0.38 (0.14) 0.36 (0.15) 0.07 (0.05) 4 Asp265Ala 1.23 (0.14) 40.16 (0.05) 9 0.07 (0.01) 0.13 (0.05) 0.09 (0.06) 4 Asn297Ala 0.80(0.18) 8 0.15 (0.06) 7 0.05 (0.00) 0.10 (0.02) 0.03 (0.01) 3 Ala327Gln0.97 0.60 (0.12) 9 0.13 (0.03) 0.14 (0.03) 0.06 (0.01) 4 Pro329Ala 0.800.48 (0.10) 6 0.08 (0.02) 0.12 (0.08) 0.21 (0.03) 4 Class 2 = Reducedbinding to FcγRII and FcγRIIIA Asp270Ala 1.05 0.76 (0.12) 6 0.06 (0.02)0.10 (0.06) 0.14 (0.04) 6 Gln295Ala 0.79 1.00 (0.11) 4 0.62 (0.20) 0.50(0.24) 0.25 (0.09) 5 Ala327Ser 0.86 (0.03) 4 0.23 (0.06) 0.22 (0.05)0.06 (0.01) 4 Class 3 = Improved binding to FcγRII and FcγRIIIAThr256Ala 1.91 (0.43) 6 1.01 (0.07) 5 1.41 (0.27) 2.06 (0.66) 1.32(0.18) 9 Lys290Ala 0.79 (0.14) 3 1.01 (0.06) 11 1.30 (0.21) 1.38 (0.17)1.31 (0.19) 9 Class 4 = Improved binding to FcγRII and no effect onFcγRIIIA Arg255Ala 0.59 (0.19) 4 0.99 (0.12) 7 1.30 (0.20) 1.59 (0.42)0.98 (0.18) 5 Glu258Ala 1.18 1.18 (0.13) 4 1.33 (0.22) 1.65 (0.38) 1.12(0.12) 5 Ser267Ala 1.08 1.09 (0.08) 10 1.52 (0.22) 1.84 (0.43) 1.05(0.24) 11 Glu272Ala 1.34 (0.24) 4 1.05 (0.06) 7 1.23 (0.12) 1.53 (0.22)0.80 (0.18) 6 Asn276Ala 1.15 (0.21) 3 1.05 (0.14) 4 1.29 (0.20) 1.34(0.40) 0.95 (0.04) 4 Asp280Ala 0.82 1.04 (0.08) 10 1.34 (0.14) 1.60(0.31) 1.09 (0.20) 10 His285Ala 0.85 0.96 (0.07) 4 1.26 (0.12) 1.23(0.15) 0.87 (0.04) 4 Asn286Ala 1.24 (0.04) 2 0.95 (0.18) 16 1.24 (0.23)1.36 (0.15) 1.05 (0.04) 6 Thr307Ala 1.81 (0.32) 6 0.99 (0.14) 4 1.07(0.15) 1.27 (0.24) 1.09 (0.18) 10 Leu309Ala 0.63 (0.18) 4 0.93 (0.18) 61.13 (0.08) 1.26 (0.12) 1.07 (0.20) 3 Asn315Ala 0.76 (0.14) 3 0.99(0.16) 6 1.15 (0.06) 1.30 (0.17) 1.07 (0.21) 5 Lys326Ala 1.03 1.03(0.05) 10 1.23 (0.20) 1.41 (0.27) 1.23 (0.23) 7 Pro331Ala 0.85 1.01(0.09) 7 1.29 (0.14) 1.34 (0.35) 1.08 (0.19) 4 Ser337Ala 1.03 1.17(0.23) 3 1.22 (0.30) 1.26 (0.06) 0.94 (0.18) 4 Ala378Gln 1.32 (0.13) 31.06 (0.05) 3 1.40 (0.17) 1.45 (0.17) 1.19 (0.17) 5 Glu430Ala 0.93(0.03) 2 1.05 (0.02) 3 1.24 (0.11) 1.28 (0.10) 1.20 (0.18) 5 Class 5 =Improved binding to FcγRII and reduced binding to FcγRIIIA His268Ala1.02 (0.22) 3 1.09 (0.11) 8 1.21 (0.14) 1.44 (0.22) 0.54 (0.12) 13Arg301Ala 0.86 1.06 (0.10) 4 1.14 (0.13) 1.29 (0.16) 0.22 (0.08) 7Lys322Ala 0.98 0.94 (0.04) 9 1.17 (0.11) 1.28 (0.21) 0.62 (0.12) 6 Class6 = Reduced binding to FcγRII and no effect on FcγRIIIA Arg292Ala 0.81(0.18) 4 0.95 (0.05) 8 0.27 (0.13) 0.17 (0.07) 0.89 (0.17) 10 Lys414Ala1.02 1.00 (0.04) 3 0.64 (0.15) 0.58 (0.18) 0.82 (0.27) 3 Class 7 =Reduced binding to FcγRII and improved binding to FcγRIIIA Ser298Ala0.80 1.11 (0.03) 9 0.40 (0.15) 0.23 (0.13) 1.34 (0.20) 16 Class 8 = Noeffect on FcγRII and reduced binding to FcγRIIIA Ser239Ala 1.06 0.81(0.09) 7 0.73 (0.25) 0.76 (0.36) 0.26 (0.08) 3 Glu269Ala 1.05 0.61(0.14) 9 0.65 (0.18) 0.75 (0.29) 0.45 (0.13) 5 Glu293Ala 0.85 1.11(0.07) 4 1.08 (0.19) 1.07 (0.20) 0.31 (0.13) 6 Tyr296Phe 0.79 1.03(0.09) 8 0.97 (0.23) 0.86 (0.17) 0.55 (0.12) 6 Val303Ala 1.26 (0.21) 30.91 (0.11) 5 0.86 (0.10) 0.65 (0.17) 0.33 (0.09) 8 Ala327Gly 0.96(0.01) 3 0.92 (0.09) 0.83 (0.10) 0.36 (0.05) 3 Lys338Ala 1.14 0.90(0.05) 3 0.78 (0.09) 0.63 (0.08) 0.15 (0.01) 2 Asp376Ala 1.45 (0.36) 41.00 (0.05) 3 0.80 (0.16) 0.68 (0.14) 0.55 (0.10) 5 Class 9 = No effecton FcγRII and improved binding to FcγRIIIA Glu333Ala 1.03 (0.01) 2 0.98(0.15) 5 0.92 (0.12) 0.76 (0.11) 1.27 (0.17) 10 Lys334Ala 1.05 (0.03) 21.06 (0.06) 11 1.01 (0.15) 0.90 (0.12) 1.39 (0.19) 16 Ala339Thr 1.06(0.04) 6 1.09 (0.03) 1.20 (0.03) 1.34 (0.09) 2 Class 10 = Effect onlyFcRn Ile253Ala 0.10 0.96 (0.05) 4 1.14 (0.02) 1.18 (0.06) 1.08 (0.14) 3Ser254Ala <0.10 0.96 (0.08) 4 0.97 (0.24) 1.15 (0.38) 0.73 (0.14) 3Lys288Ala 0.38 (0.12) 5 0.88 (0.15) 15 1.15 (0.26) 1.14 (0.20) 1.06(0.04) 4 Val305Ala 1.46 (0.48) 6 1.04 (0.19) 10 1.12 (0.12) 1.23 (0.22)0.84 (0.15) 4 Gln311Ala 1.62 (0.25) 4 0.93 (0.05) 4 1.11 (0.06) 1.19(0.13) 0.93 (0.17) 3 Asp312Ala 1.50 (0.06) 4 1.01 (0.12) 5 1.20 (0.24)1.19 (0.07) 1.23 (0.14) 3 Lys317Ala 1.44 (0.18) 4 0.92 (0.17) 6 1.13(0.05) 1.18 (0.27) 1.10 (0.23) 4 Lys360Ala 1.30 (0.08) 4 1.02 (0.04) 31.12 (0.10) 1.12 (0.08) 1.23 (0.16) 6 Gln362Ala 1.25 (0.24) 3 1.00(0.04) 3 1.03 (0.10) 1.02 (0.03) 1.03 (0.16) 4 Glu380Ala 2.19 (0.29) 61.04 (0.06) 3 1.18 (0.01) 1.07 (0.05) 0.92 (0.12) 2 Glu382Ala 1.51(0.18) 4 1.06 (0.03) 3 0.95 (0.11) 0.84 (0.04) 0.76 (0.17) 3 Ser415Ala0.44 1.04 (0.03) 3 0.90 (0.11) 0.88 (0.05) 0.86 (0.18) 2 Ser424Ala 1.41(0.14) 3 0.98 (0.03) 3 1.04 (0.06) 1.02 (0.02) 0.88 (0.09) 2 His433Ala0.41 (0.14) 2 0.98 (0.03) 3 0.92 (0.18) 0.79 (0.18) 1.02 (0.15) 3Asn434Ala 3.46 (0.37) 7 1.00 (0.04) 3 0.96 (0.06) 0.97 (0.12) 0.77(0.13) 6 His435Ala <0.10 4 1.25 (0.09) 3 0.77 (0.05) 0.72 (0.05) 0.78(0.03) 3 Tyr436Ala <0.10 2 0.99 (0.02) 2 0.93 (0.05) 0.91 (0.06) 0.91(0.15) 3 ^(a)Residue numbers are according to the Eu numbering system(Kabat et al. (1991)). Variants that had no effect on binding (i.e. didnot reduce binding by more than 60% or improve binding by more than 20%)to FcγR or FcRn were: Lys246, Lys248, Asp249, Met252, Thr260, Lys274,Tyr278, Val282, Glu283, Thr289, Glu294, Tyr300Phe, Glu318, Lys320,Ser324, Ala330Gln, Thr335, Lys340, Gln342, Arg344, Glu345, Gln347,Arg355, Glu356, Met358, Thr359, Lys360, Asn361, Tyr373, Ser375, Ser383,Asn384, Gln386, Glu388, Asn389, Asn390, Tyr391Phe, Lys392, Leu398,Ser400, Asp401, Asp413, Arg416, Gln418, Gln419, Asn421, Val422, Thr437,Gln438, Lys439, Ser440, Ser442, Ser444, Lys447. ^(b)Values are the ratioof binding of the variant to that of native IgG1 at 0.33 or 1 μg/ml. Avalue greater than 1 denotes binding of the variant was improvedcompared to native IgG1 while a ratio less than 1 denotes reducedbinding compared to native IgG1. Reduced binding to any given receptorwas defined as a reduction of ≧40% compared to native IgG; betterbinding was defined as an improvement of ≧25% compared to native IgG1.^(c)Number of independent assays for FcγRIIA, FcγRIIB and FcγRIIIA. Atleast two separately expressed and purified lots of each variant wereassayed.

Aside from alanine variants, various non-alanine substitution variantswere made, and the FcR binding activity of those variants is summarizedin the following table.

TABLE 7 NON-ALANINE VARIANTS Res#EU FcRn FcγRI FcγRIIA^(a) FcγRIIBFcγRIIIA IG2 (Kabat) mean sd n mean sd n mean sd mean sd mean sd 222D249(262)E 0.97 0.99 0.84 n = 1^(b) 176 T256(269)G 1.10 (0.03) 1.06(0.07) 0.96 (0.27) n = 2 254 T256(269)N 1.03 0.89 1.13 n = 1 157D265(278)N 0.02 (0.01) 0.03 (0.01) 0.02 (0.01) n = 3 158 D265(278)E 0.11(0.04) 0.03 (0.01) 0.02 (0.01) n = 3 189 S267(280)G R131 1.18 (0.10)0.95 (0.16) 0.08 (0.02) n = 4 H131 0.59 (0.09) n = 3 84 H268(281)N 1.331.41 0.56 n = 1 85 H268(281)S 1.35 1.38 0.81 n = 1 87 H268(281)Y 1.191.29 0.76 n = 1 168 E269(282)D 0.89 (0.10) 0.73 (0.07) 1.13 (0.21) n = 2169 E269(282)Q 0.08 (0.01) 0.16 (0.00) 0.28 (0.03) n = 2 92 D270(283)N0.03 (0.02) 0.05 (0.05) 0.04 (0.03) n = 5 93 D270(283)E 0.08 (0.01) 0.06(0.01) 0.90 (0.17) n = 3 223 E272(285)Q 1.93 1.81 0.82 n = 1 224E272(285)N 0.43 0.23 0.50 n = 1 167 K274(287)Q 0.86 0.94 0.62 n = 1 165N276(289)K 0.81 0.77 0.61 n = 1 233 N276(289)Q 1.09 0.79 0.91 n = 1 79D280(295)N 1.07 (0.18) 1.22 (0.19) 1.16 (0.21) n = 6 149 D280(295)S 1.07(0.06) 1.04 (0.08) 1.09 (0.06) n = 2 226 E283(300)Q 1.12 1.24 1.19 n = 1227 E283(300)S 1.03 1.07 0.85 n = 1 228 E283(300)N 1.18 1.28 0.94 n = 1229 E283(300)D 1.14 1.23 0.95 n = 1 23 N286(303)Q 1.52 1.13 0.96 n = 1237 N286(303)S 1.72 1.38 1.32 n = 1 238 N286(303)D 1.41 1.23 0.98 n = 173 K290(307)Q 1.17 1.26 1.40 n = 1 75 K290(307)S 1.27 1.34 1.26 n = 1 77K290(307)E 1.14 1.10 1.20 1.30 n = 1 78 K290(307)R 1.25 1.05 1.15 1.08 n= 1 177 K290(307)G 1.07 1.21 1.23 n = 1 80 R292(309)K 0.71 (0.17) 0.75(0.10) 1.15 (0.18) n = 3 81 R292(309)H 0.21 (0.09) 0.12 (0.01) 0.92(0.08) n = 2 82 R292(309)Q 0.47 (0.12) 0.25 (0.06) 0.45 (0.09) n = 3 83R292(309)N 0.54 (0.16) 0.29 (0.07) 0.88 (0.02) n = 3 144 E293(310)Q 0.85(0.03) 0.77 (0.13) 0.99 (0.04) n = 2 145 E293(310)D 0.90 (0.02) 0.88(0.07) 0.37 (0.07) n = 3 147 E293(310)K 1.13 (0.04) 1.31 (0.17) 0.72(0.08) n = 4 173 E294(311)Q 1.01 0.95 0.84 n = 1 174 E294(311)D 0.370.26 0.14 n = 1 185 Y296(313)H 0.90 0.81 0.92 n = 1 186 Y296(313)W 0.960.93 1.38 n = 1 70 S298(317)G 0.87 (0.17) 0.63 (0.33) 0.46 (0.09) n = 471 S298(317)T 0.29 (0.19) 0.27 (0.19) 0.73 (0.21) n = 6 72 S298(317)N0.05 (0.03) 0.08 (0.08) 0.06 (0.03) n = 5 218 S298(317)V 0.11 (0.06)0.17 (0.01) 0.33 (0.19) n = 3 219 S298(317)L 1.14 (0.12) 1.42 (0.31)0.34 (0.04) n = 3 150 V303(322)L 0.89 (0.05) 0.73 (0.10) 0.76 (0.09) n =4 151 V303(322)T 0.64 (0.11) 0.34 (0.05) 0.20 (0.05) n = 4 217E318(337)K 1.03 1.08 0.72 n = 1 172 K320(339)R 0.71 0.66 0.68 n = 1 202K320(339)M 1.34 1.40 1.27 n = 1 204 K320(339)Q 1.23 1.12 1.17 n = 1 205K320(339)E 1.29 1.34 1.12 n = 1 235 K320(339)R 1.24 0.95 0.86 n = 1 155K322(341)R 0.87 (0.07) 0.87 (0.21) 0.92 (0.15) n = 3 156 K322(341)Q 0.87(0.02) 0.92 (0.23) 0.78 (0.18) n = 3 206 K322(341)E 1.38 1.34 0.81 n = 1207 K322(341)N 0.57 0.36 0.04 n = 1 213 S324(343)N 1.15 1.09 0.97 n = 1214 S324(343)Q 0.82 0.83 0.78 n = 1 215 S324(343)K 0.66 0.37 0.77 n = 1216 S324(343)E 0.82 0.73 0.81 n = 1 208 K326(345)S 1.44 1.62 1.37 n = 1209 K326(345)N 1.04 1.00 1.27 n = 1 210 K326(345)Q 1.36 1.41 1.15 n = 1211 K326(345)D 1.68 2.01 1.36 n = 1 212 K326(345)E 1.34 (0.27) 1.47(0.33) 1.26 (0.04) n = 2 131 A327(346)S 0.23 (0.06) 0.22 (0.05) 0.06(0.01) n = 4 159 A327(346)G 0.92 (0.09) 0.83 (0.10) 0.36 (0.05) n = 3196 A330(349)D 0.18 0.08 0.07 n = 1 197 A330(349)K 1.28 1.25 1.28 n = 1198 P331(350)S 0.91 (0.08) 0.78 (0.07) 0.58 (0.19) n = 4 199 P331(350)N0.86 0.65 0.23 n = 1 200 P331(350)E 1.06 0.91 0.42 n = 1 203 P331(350)K0.94 0.71 0.33 n = 1 141 E333(352)Q 0.70 (0.05) 0.64 (0.09) 1.05 (0.09)n = 3 142 E333(352)N 0.59 (0.04) 0.52 (0.07) 0.56 (0.10) n = 4 143E333(352)S 0.94 n = 1 152 E333(352)K 0.85 (0.14) n = 3 153 E333(352)R0.75 (0.04) 0.66 (0.03) 0.84 (0.05) n = 2 154 E333(352)D 1.26 (0.04) n =3 178 E333(352)G 0.87 0.76 1.05 n = 1 179 K334(353)G 0.76 (0.08) 0.60(0.13) 0.88 (0.22) n = 5 135 K334(353)R 1.15 (0.09) 1.33 (0.18) 0.68(0.07) n = 5 136 K334(353)Q 1.08 (0.11) 1.10 (0.21) 1.23 (0.08) n = 7137 K334(353)N 1.16 (0.11) 1.29 (0.30) 1.11 (0.12) n = 8 138 K334(353)S1.01 (0.11) 1.03 ().05) 1.19 (0.08) n = 3 139 K334(353)E 0.74 (0.15)0.72 (0.12) 1.30 (0.09) n = 6 140 K334(353)D 0.51 (0.09) 0.40 (0.03)1.13 (0.09) n = 4 190 K334(353)M 1.18 0.99 (0.13) 0.93 (0.15) 0.49(0.04) n = 2 191 K334(353)Y 1.15 1.08 1.05 1.31 n = 1 192 K334(353)W1.16 0.94 0.91 1.07 n = 1 193 K334(353)H 1.11 1.09 1.07 1.26 n = 1 220K334(353)V 1.13 (0.11) 1.09 (0.15) 1.34 (0.18) n = 3 221 K334(353)L 1.051.09 1.38 n = 1 171 T335(354)Q 0.86 0.79 0.84 n = 1 194 T335(354)E 1.241.30 1.19 n = 1 195 T335(354)K 1.19 1.14 1.30 n = 1 273 A339(359)T 1.231.11 1.23 1.42 n = 1 S267(280)T 0.42 (0.10) 0.45 (0.01) 0.05 (0.05) n =3 R301(320)M 1.29 (0.17) 1.56 (0.12) 0.48 (0.21) n = 4 K338(358)M 0.99(0.13) 0.93 (0.15) 0.49 (0.04) n = 2 ^(a)Values are the ratio of bindingof the variant to that of native IgG1 at 0.33 or 1 μg/ml. A valuegreater than 1 denotes binding of the variant was improved compared tonative IgG1 while a ratio less than 1 denotes reduced binding comparedto native IgG1. ^(b)Number of independent assays for FcγRIIA, FcγRIIBand FcγRIIIA. At least two separately expressed and purified lots ofeach variant were assayed.

The following table summarizes the FcR binding activity of variouscombination variants.

TABLE 8 COMBINATION VARIANTS Res#EU FcRn^(a) FcγRI FcγRIIA^(b) FcγRIIBFcγRIIIA IG2 (Kabat) mean sd n mean sd n mean sd mean sd mean sd 96S267(280)A 1.41 (0.00) 1.56 (0.16) 0.96 (0.12) n = 2 H268(281)A 134E333(352)A 0.72 (0.08) 0.63 (0.13) 1.30 (0.12) n = 5 K334(353)A 1059T256(269)A 0.44 (0.03) 0.22 (0.04) 1.41 (0.06) n = 2 S298(317)A 1051T256(269)A 0.47 (0.01) 0.30 (0.03) 1.21 (0.26) n = 2 D280(295)AS298(317)A T307(326)A 106 T256(269)A 0.11 0.08 0.90 n = 1 D280(295)AR292(309)A S298(317)A T307(326)A 107 S298(317)A 0.34 (0.05) 0.16 (0.08)1.53 (0.24) n = 5 E333(352)A 109 S298(317)A 0.41 (0.07) 0.19 (0.08) 1.62(0.34) n = 6 K334(353)A 110 S298(317)A 0.34 (0.15) 0.15 (0.06) 1.51(0.31) n = 10 E333(352)A K334(353)A 246 S267(280)A 1.62 (0.15) 2.01(0.45) 1.04 (0.12) n = 2 E258(271)A 247 S267(280)A 1.60 (0.18) 1.72(0.13) 0.88 (0.07) n = 3 R255(268)A 248 S267(280)A 1.54 (0.08) 1.96(0.37) 1.13 (0.07) n = 2 D280(295)A 250 S267(280)A 1.51 (0.13) 1.82(0.32) 0.95 (0.05) n = 3 E272(285)A 251 S267(280)A 1.67 (0.11) 1.85(0.10) 0.92 (0.09) n = 3 E293(310)A 264 S267(280)A 1.48 (0.12) 2.03(0.30) 0.89 (0.04) n = 2 E258(271)A D280(295)A R255(268)A 269 E380(405)A8.0 (1.0) 6 1.02 (0.07) 1.05 (0.11) 1.02 n = 2 N434(465)A 270 E380(405)A11.8 (1.5) 5 0.99 (0.06) 0.99 (0.11) 0.96 n = 2 N434(465)A T307(326)A271 E380(405)A 0.9 (0.1) 4 0.98 1.04 0.92 n = 1 L309(328)A 272N434(465)A 2.9 (0.4) 4 0.94 (0.11) 0.96 (0.17) 0.88 n = 2 K288(305)A^(a)Values are the ratio of binding of the variant to that of nativeIgG1 at pH 6.0. ^(b)Values are the ratio of binding of the variant tothat of native IgG1 at 0.33 or 1 μg/ml. A value greater than 1 denotesbinding of the variant was improved compared to native IgG1 while aratio less than 1 denotes reduced binding compared to native IgG1.

DISCUSSION

This study includes a complete mapping of human IgG1 for human FcγRI,FcγRIIA, FcγRIIB, FcγRIIIA, and FcRn. An alanine-scan of all amino acidsin human IgG1 Fc (CH2 and CH3 domains) exposed to solvent, based on thecrystal structure of human Fc (Deisenhofer, Biochemistry 20:2361-2370(1981)), was performed. Each exposed amino acid in CH2 and CH3 wasindividually changed to alanine and the variant IgG assayed against allfive human receptors; all variants were evaluated using humanizedanti-IgE E27 IgG1 as the parent polypeptide. FcγRI and FcRn are highaffinity receptors and monomeric IgG could be evaluated in the assaysfor these two receptors. FcγRIIA, FcγRIIB and FcγRIIIA are low affinityreceptors and required use of an immune complex. Hence, an ELISA-typeassay was used for FcγRIIA, FcγRIIB, and FcγRIIIA, in which pre-formedhexamers, consisting of three anti-IgE E27 and three IgE molecules werebound to the FcγR and either anti-human IgG Fc-HRP or protein G-HRP usedas detection reagent. In order to increase binding, these hexamers couldbe linked into multimers by addition of human VEGF (using anti-VEGFIgE). The hexamers bound to the low affinity FcγR significantly betterthan the IgG monomers; the multimers bound better than the hexamers(FIGS. 15A and 15B). The hexameric complexes were used since theseprovided sufficient binding and required less IgG. Complexes formedusing other antibody:antigen combinations are also possible reagents, aslong as the antigen contains at least two identical binding sites permolecule for the antibody. As an example, VEGF contains two bindingsites per VEGF dimer for anti-VEGF A.4.6.1 (Kim et al., Growth Factors7:53 (1992) and Kim et al. Nature 362:841 (1993)). VEGF:anti-VEGFmultimers also bound to the low affinity FcγRIIA and FcγRIIIA (FIGS. 16Aand 16B).

Classes of Fc Region Variants

Once the complete alanine-scan was performed, several classes of alaninevariants were found. Some variants exhibited reduced binding to all FcγR(G14 D265A; FIG. 17), while other variants showed reduced binding onlyto one FcγR (G16 H268A; FIG. 17), improved binding only to one FcγR (G15S267A; G54 E333A; G55 K334A; FIG. 17), or simultaneous reduction to oneFcγR with improvement to another (G36 S298A; FIG. 17).

The IgG1 variants can be separated into distinct classes based on theireffects on binding to the various receptors. Class 1 consists ofvariants which showed reduced binding to all FcγR (Table 6) and areclustered near the region of the CH2 domain where the hinge joins CH2(FIG. 26A). In the Glu233Pro/Leu234Val/Leu235Ala/Gly236 deleted variant,part of the so-called lower hinge region (residues 233-239) of humanIgG1 was exchanged with that of human IgG2. The reduction in binding toall FcγR is in agreement with previous studies; this variant also showedimpaired binding to FcRn. Two other residues in the lower hinge regionwere individually changed to Ala; Pro238Ala (Class 1) had a morepronounced effect than Ser239Ala (Class 8). If the Pro238Ala effect isdue to a conformation change, this change was beneficial for binding toFcRn. In contrast, Pro329Ala showed a relatively modest reduction inbinding to FcRn compared to the significant reduction in binding to theFcγR. In the IgG1 Fc:FcγRIIIA crystal structure (Sondermann et al.Nature 406:267-273 (2000)), Pro329 intimately interacts with two Trpsidechains of the receptor and the loss of these interactions byPro329Ala may account for the severe reduction in binding. Pro329 isalso involved in binding of human IgG1 to human C1q (Example 2 herein).

Removal of the conserved Asn-linked glycosylation site in the CH2domain, Asn297Ala, abolished binding, in agreement with earlier studies(Lund et al. J. Immunol. 157: 4963-4969 (1996); and Lund et al. FASEB J.9: 115-119 (1995)). Another residue which interacts with carbohydrate,Asp265, has also been previously found to be important in human IgG3binding to human FcγRI (Lund et al. J. Immunol. 157: 4963-4969 (1996);and Lund et al. FASEB J. 9: 115-119 (1995)). In human IgG1, changingAsp265 to Ala, Asn or Glu nullified binding (Tables 6 and 7), suggestingthat both the charge and size are important. The results of theAla327Gln (Class 1) and Ala327Ser (Class 2) variants imply that theregion around Ala327 involved in binding to the FcγR may require a closefit between receptor and IgG1, as enlarging this sidechain diminishedbinding. This position is an Asp in mouse IgG2a and IgG2b and thereforechanging the human IgG1 Ala to a larger sidechain is unlikely to haveaffected the conformation. The Ala327Gly (Class 8) variant reducedbinding only to FcγRIIIA suggesting that this receptor requires thepresence of a small amino acid sidechain at this position whereas theother receptors do not.

Class 1 variants (in addition to the hinge residues, which were notinvestigated in this study) comprise the entire binding site on IgG1 forFcγRI. Residues in the F(ab) portions of the IgG1 do not contribute toFcγRI binding as evidenced by both a CD4-immunoadhesin (Capon et al.Nature 337:525-531 (1989)) and an Fc fragment binding to FcγRI aseffectively as did intact IgG1. Notably, no variants were found whichreduced binding only to FcγRI.

Class 2 consists of three variants with reduced binding to FcγRII andFcγRIII but not FcγRI. Like the residues in Class 1, Asp270 and Gln295are located near the hinge (FIG. 26A). In crystal structures of humanIgG1 Fc (Deisenhofer J., Biochemistry 20: 2361-2370 (1981); Guddat etal., PNAS(USA) 90: 4271-4275 (1993)), Gln295 is completely solventexposed whereas Asp270, though exposed, forms hydrogen bonds from itssidechain OS atom to the backbone nitrogens of Lys326 and Ala327 and tothe sidechain N6 of Asn325. Disruption of these interactions byAsp270Ala could cause a local conformational perturbation that effectedthe severe reduction in binding to FcγRII and FcγRIIIA. However,Asp270Ala did not affect binding to FcγRI or FcRn. Furthermore,Asp270Asn, which could maintain the aforementioned hydrogen bonds, alsoabolished binding to FcγRII and FcγRIIIA, and Asp270Glu bound toFcγRIIIA as effectively as did native IgG1 (Table 7). Taken together,these data suggest that the sidechain charge of Asp270 is important forinteraction with FcγRII and FcγRIIIA.

Class 3 consists of two variants with improved binding to FcγRIIA,FcγRIIB, and FcγRIIIA. Thr256 and Lys290 are located near one another inthe CH2 domain (FIG. 26). Thr256Ala also exhibited improved binding toFcRn.

Class 4 variants were characterized by improved binding only to FcγRIIAand FcγRIIB. Those that improved binding to FcγRII the most—Arg255Ala,Glu258Ala, Ser267Ala, Glu272Ala, and Asp280Ala—are distant from oneanother in the CH2 domain (FIG. 26). Of these, only Ser267 was cited asan interacting residue in the Fc:FcγRIIA crystal structure (Sondermannet al. Nature 406:267-273 (2000)). Ser267Ala improved binding only toFcγRII, Ser267Gly abolished binding only to FcγRIIIA and Ser267Thrreduced binding to FcγRII and FcγRIIIA (Tables 6 and 7). Asp280Asn(Table 7), like Asp280Ala (Table 6), improved binding only to FcγRII.Three of the Class 4 residues also exhibited improved binding to FcRn(Glu272Ala, Thr307Ala, Ala378Gln) while Arg255Ala exhibited reducedbinding. Ala378 interacts with CH2 domain loop AB, which containsresidues that interact directly with FcRn, and hence may influencebinding to FcRn indirectly.

Class 5 variants exhibited improved binding to FcγRIIA and FcγRIIB but,in contrast to Class 4, also showed reduced binding to FcγRIIIA. Ofthese, Lys322 has also been implicated in human C1q binding (Example 2above). The aliphatic portion of the Arg301 sidechain is buried andinteracts with the Tyr296 sidechain, at least in some crystalstructures, while the Arg301 guanidinium group may interact with theAsn297-linked carbohydrate (Deisenhofer, J. Biochemistry 20: 2361-2370(1981); Guddat et al. Proc. Natl. Acad. Sci. USA 90: 4271-4275 (1993);and Harris et al. J. Mol. Biol. 275: 861-872 (1998)). The Arg301Alavariant effected a modest improvement in binding to FcγRIIB and apronounced reduction in binding to FcγRIIIA (Table 6); the Arg301Metvariant, which may maintain the aliphatic interaction of the Arg301sidechain, showed improved binding to FcγRIIA and FcγRIIB and a lesspronounced reduction of binding to FcγRIIIA compared to Arg301Ala (Table7).

Class 6 residues show diminished binding to FcγRII only. Arg292Ala islocated in the CH2 domain distant from the hinge. Lys414 is at the“bottom” of the IgG1, spatially removed from all other residues havingan effect on FcγRII binding, suggesting that it may play only a minorrole in binding (discussed below).

Class 7 is comprised of Ser298Ala which reduced binding to FcγRII, butimproved binding to FcγRIIIA. Situated among the Class 1 residues nearthe hinge (FIG. 26), Ser298 is also part of the Asn-linked glycosylationsequence Asn297-Ser298-Thr299. Ser298Thr followed the pattern ofSer298Ala, whereas Ser298Asn abolished binding to FcγRIIIA as well asFcγRII (Table 7).

Reduced binding only to FcγRIIIA characterizes Class 8 and includes fiveresidues in the CH2 domain and two in the CH3 domain (Ala327Gln is inClass 1). Ser239 has been previously identified as playing a minor rolein murine IgG2b binding to murine FcγRII (Lund et al. Mol. Immunol. 29:53-59 (1992)) and in the IgG1 Fc:FcγRIIIA crystal structure (Sondermannet al. Nature 406:267-273 (2000)), the Ser239 in one of the two heavychains forms a hydrogen-bond to the Lys117 sidechain of FcγRIIIA. Incontrast, Glu293Ala (Table 6) and Glu293Asp (Table 7) reduced binding asmuch as did Ser239Ala even thought Glu293 is not located near theFc:FcγRIIIA interface in the crystal structure. In some crystalstructures (Deisenhofer, J. Biochemistry 20: 2361-2370 (1981); Guddat etal. Proc. Natl. Acad. Sci. USA 90: 4271-4275 (1993); and Harris et al.J. Mol. Biol. 275: 861-872 (1998)), the Tyr296 sidechain interactsintimately with the aliphatic portion of Arg301 (Class 5); alteringeither of these reduced binding to FcγRIIIA. Note, however, that Tyr296was changed to Phe (not Ala) and the 50% reduction in binding toFcγRIIIA is believed to be due to removal of the sidechain hydroxylgroup.

At position Lys338, altering the sidechain to Ala or Met effectedreduction in binding to FcγRIIIA, suggesting that both the sidechaincharge and aliphatic portions are required. The Lys338 sidechain formspart of the interface between the CH2 and CH3 domains and participatesin a salt-bridge with Glu430 in several crystal structures (Deisenhofer,J. Biochemistry 20: 2361-2370 (1981); Harris et al. J. Mol. Biol. 275:861-872 (1998); Harris et al. Biochemistry 36: 1581-1597 (1997)) (FIG.26B). While it is possible that altering Lys338 may disrupt the CH2:CH3interface and thereby influence binding, Lys338Ala (Table 6) andLys338Met (Table 7) did not disrupt binding to FcγRI, FcγRII or FcRn.Since it is known that binding of IgG1 to FcRn involves residues in bothCH2 and CH3 (Raghavan and Bjorkman Annu. Rev. Cell Dev. Biol. 12:181-220 (1996); Ward and Ghetie Ther. Immunol. 2: 77-94 (1995); andBurmeister et al. Nature 372: 379-383 (1994)), this suggests that anyconformational effect of Lys338Ala must be local and minimal. Note alsothat while Lys338Ala and Lys338Met reduced binding to FcγRIIIA,Glu430Ala (Class 4) improved binding, suggesting that the Lys338:Glu430salt-bridge is not essential in maintaining binding. Another CH3 residueaffecting FcγRIIIA is Asp376 that interacts with the CH2 domain.

Class 9 is characterized by improved binding only to FcγRIIIA andincludes Glu333Ala, Lys334Ala and Ala339Thr. A previous study found thatAla339Thr improved binding to FcγRI (Chappel et al. J. Biol. Chem. 268:25124-25131 (1993)); in this study the Ala339Thr variant bound betterthan native IgG1 to FcγRIIIA but not FcγRI (Table 6). Several non-Alavariants were tested at Glu333 and Lys334. Glu333Asp also improvedbinding to FcγRIIIA while Glu333Asn reduced binding to FcγRII as well asFcγRIIIA (Table 7). At position 334, changing Lys to Gln, Glu or Valmaintained the improved binding to FcγRIIIA (Table 7). Surprisingly, theLys334Arg variant reversed the receptor preference, i.e. this variantbound better to FcγRIIB, not FcγRIIIA as for the Lys334Ala variant.Taken together these data suggest that FcγRIIIA interacts with Lys334even though this residue is not among the IgG1 residues found tointeract with FcγRIIIA in the co-crystal structure (Sondermann et al.Nature 406:267-273 (2000)).

Class 10 residues influenced binding only to FcRn. Note that residues inother classes may also have affected binding to FcRn, but wereclassified according to their effect on FcγR. Positions whicheffectively abrogated binding to FcRn when changed to alanine includeIle253, Ser254, His435, and Tyr436. Other positions showed a lesspronounced reduction in binding: Glu233-Gly236 (Class 1), Arg255 (Class4), Lys288, Ser415, and His433. Several amino acid positions exhibitedan improvement in FcRn binding when changed to alanine; notable amongthese are Pro238 (Class 1), Thr256 (Class 3), Thr307 (Class 4), Gln311,Asp312, Glu380, Glu382 and Asn434. The pattern of binding was the samewhen a second assay format was used, e.g. with IgE-coated plates ratherthan FcRn-coated plates.

The set of IgG1 residues involved in binding to all human FcγR arerepresented by Class 1 (Table 6). Indeed, this set comprises the entirebinding site on IgG1 for FcγRI. Class 1 residues are located in the CH2domain proximal to the hinge and fall into two categories: (1) positionsthat may interact directly with all FcγR include Leu234-Pro238, Ala327,and Pro329 (and possibly Asp265); (2) positions that influencecarbohydrate nature or position include Asp265 and Asn297.

Previous studies mapping the binding residues in mouse or human IgG haveconcentrated primarily on the lower hinge region, i.e. residuesLeu234-Ser239, revealing Leu234 and Leu235 as the two most important forFcγRI (Duncan et al. Nature 332, 563-564 (1988); Canfield and MorrisonJ. Exp. Med. 173:1483-1491 (1991)) and Leu234 and Gly237 as the two mostimportant for FcγRII (Lund et al. Mol. Immunol. 29:53-59 (1992);Sondermann et al. Nature 406: 267-273 (2000)). Of the two residues inthe lower hinge investigated in this study, Pro238Ala affected bindingto all FcγR while Ser239Ala affected binding only to FcγRIIIA.

In the co-crystal structure of IgG1 Fc:FcγRIIIA (Sondermann et al.Nature 406: 267-273 (2000)), Pro329 interacts with two Trp sidechainsfrom the receptor and a similar interaction may occur with the otherFcγR. However, removal of the Pro sidechain, as in Pro329Ala, mightcause a localized conformational change which perturbs adjacent bindingresidues, supported by the above report that Pro329Ala also effects C1qbinding (Example 2). Ala327Gln could be causing steric hindrance tobinding due to introduction of a large sidechain at this position,though altering Ala327 to Ser did not affect binding to FcγRI.Inspection of the IgG1 Fc:FcγRIIIA crystal structure shows that Ala327is near the IgG1:FcγRIIIA interface and forms a van der Waals'interaction with the Trp87 sidechain; however, it is not obvious whyintroduction of a larger sidechain such as Ser or Gln should so severelyreduce binding. For Asn297 and Asp265, earlier studies evaluated therequirement for carbohydrate attached at Asn297 as well as the influenceof Asp265 on the nature of the carbohydrate (Lifely et al. Glycobiology5:813-822 (1995); Lund et al. J. Immunol. 157: 4963-4969 (1996); andLund et al. FASEB J. 9: 115-119 (1995)); these will be discussed below.

For FcγRI the IgG segment Gly316-Ala339 has also been previouslyimplicated based on sequence comparison and binding of IgG subclassesfrom different species (Woof et al. Mol. Immunol. 23: 319-330 (1986);Burton et al. Mol. Immunol. 25: 1175-1181 (1988)) and mutagenesis(Canfield and Morrison J. Exp. Med. 173: 1483-1491 (1991); Chappel etal. J. Biol. Chem. 268: 25124-25131 (1993)). Within the segmentGly316-Ala339, however, only Ala327Gln and Pro329Ala affected binding toFcγRI (Class 1). All other exposed residues in the 316-339 segment hadno effect. In contrast to a previous study in which changing residue 331from Pro to Ser in human IgG3 reduced binding by 10-fold (Canfield andMorrison J. Exp. Med. 173: 1483-1491 (1991)), in human IgG1 thePro331Ala (Table 6) and Pro331Ser (Table 7) variants had no effect.Another previous report showed that an Ala339Thr substitution couldimprove binding to FcγRI by 3-fold (Chappel et al. J. Biol. Chem. 268:25124-25131 (1993)); in this study the Ala339Thr variant was onlyequivalent in binding to native IgG1 (Class 3).

It has been noted that the presence of the γ-chain may augment thebinding affinity of the FcγRI α-chain (Miller et al. J. Exp. Med. 183:2227-2233 (1996)). Since it is conceivable that some residues in thehuman IgG1 might interact directly with the γ-chain, binding of the IgG1variants to FcγRI on THP-1 cells was tested as well to the FcγRI α-chaincoated on a plate. The results of the two assay formats were the samefor the entire panel of variants, suggesting that the γ-chain augmentsbinding by the α-chain through a mechanism other than direct interactionwith the IgG1.

Since FcγRI binds monomeric IgG1 about 100-fold more strongly than doFcγRII and FcγRIII, one might expect that FcγRI would utilize eitherdifferent or additional IgG1 residues to effect the tighter binding.However, the set of IgG1 residues that control binding to FcγRI are asubset of those effecting binding to FcγRII and FcγRIII (Class 1). Thissuggests that the comparatively strong binding of IgG1 to FcgRI resultsfrom either (1) utilization of only two Ig-like domains of FcγRI(analogous to the two Ig-like domains of FcγRII and FcγRIII) but withinteraction of different amino acids on FcγRI than are used by FcγRIIand FcγRIII, (2) utilization of the same amino acids on all threereceptors but with additional direct interaction of amino acids in thethird FcγRI domain, or (3) the third domain of FcγRI effects aconformational change in the other two Ig-like domains which results inmore efficacious interaction of these domains with the common set ofbinding residues on IgG1. In both human and murine FcγRI, removal of thethird domain reduces affinity for monomeric IgG and alters specificityfor IgG subclasses (Hulett et al. J. Immunol. 147: 1863-1868 (1991);Porges et al. J. Clin. Invest. 90: 2102-2109 (1992)). This wouldsupport, but does not discriminate between, possibilities 2 and 3.

In contrast to FcγRI, FcγRII requires the presence of two identical IgGheavy chains (Haagen et al. J. Immunol. 154: 1852-1860 (1995)),suggesting that residues from both heavy chains may form the FcγRIIbinding site in IgG. The set of IgG1 residues, in addition to the commonClass 1 residues, which affect binding to FcγRII are: (largest effect)Arg255, Thr256, Glu258, Ser267, Asp270, Glu272, Asp280, Arg292, Ser298and (less effect) His268, Asn276, His 285, Asn286, Lys290, Gln295,Arg301, Thr307, Leu309, Asn315, Lys322, Lys326, Pro331, Ser337, Ala339,Ala378, Lys414.

A previous study elucidated the residues in murine IgG2b involved inbinding to murine FcγRII (Lund et al. Mol. Immunol. 29: 53-59 (1992)).Of the residues investigated in that study, only Asn297Ala and Glu318Alashowed a complete abrogation of binding. Several other murine IgG2bresidues exhibited more modest reduction in binding to FcγRII:Ser239Ala, Lys248Ala, Ser267Ala, Lys322Ala, Glu333Ala, Thr335Ala,Ser337Ala, and Lys340Ala (Lund et al. Mol. Immunol. 29: 53-59 (1992)).Several of these residues also exhibited modest reduction in binding inthe human system (e.g. Ser239Ala, Thr335Ala) or modest improvement inbinding (e.g. Lys340Ala) but fell outside of the cutoff used in thisstudy. Noteworthy differences between the two systems are: Asp270Alaaffecting only the human system, Glu318Ala affecting only the murinesystem, and Lys322Ala, Ser267Ala, and Ser337Ala exhibiting improvedbinding in the human system but slightly reduced binding in the murine.

In contrast to FcγRI, several variants exhibited improved binding toFcγRIIA and FcγRIIB (Classes 3, 4 and 5). Of special interest are Class4 containing residues which, when changed to Ala, improved binding onlyto FcγRII and Class 5 containing residues which, when changed to Ala,simultaneously improved binding to FcγRII and reduced binding toFcγRIIIA. These can be used to make IgG1 with improved specificity forFcγRII over FcγRIIIA.

Recently the crystal structures of human FcγRIIA (Maxwell et al. NatureStruct. Biol. 6: 437-442 (1999)) and FcγRIIB (Sondermann et al. EMBO J.18: 1095-1103 (1999)) have been solved. In the FcγRIIA report, it wassuggested that in addition to the lower hinge (Leu234-Gly237), residuesin IgG CH2 domain loops FG (residues 326-330) and BC (residues 265-271)might play a role in binding, though it was noted that these had yet tobe demonstrated by mutagenesis. Of the four exposed residues in loop FG,Ala330Gln had no effect, Ala327Gln, Ala327Ser and Pro329Ala reducedbinding, while Lys326Ala improved binding. Of the five exposed residuesin loop BC, two reduced binding when altered to Ala (Asp265Ala andAsp270Ala) and two improved binding (Ser267Ala and His268Ala). Severalof the residues found to influence binding to FcγRII lie outside of theresidues at the Fc:FcγRIIIA interface in the co-crystal structure(Sondermann et al. EMBO J. 18: 1095-1103 (1999)). One of these, Asp280,is not only distant from the Fc:FcγRIIIA interface but is distant fromother FcγRII-influencing residues (FIG. 26A). However, both Asp280Alaand Asp280Asn improved binding to FcγRII, suggesting that this residuedoes indeed interact with FcγRII.

In addition to the Class 1 residues, positions which reduced binding toFcγRIIIA by 40% or more (when changed to Ala) are: Ser239, Ser267 (Glyonly), His268, Glu293, Gln295, Tyr296, Arg301, Val303, Lys338, Asp376.In the Fc crystal structure, these residues separate into two groups.Lys338 and Asp376 are at the CH2-CH3 interface and may affect thespatial relationship of these two domains, thereby affecting FcγRIIIAbinding; note, though, that changing these two residues did notsignificantly reduce binding to FcγRI, FcγRII or FcRn. The other eightpositions are clustered together near the Class 1 residues at thehinge-proximal end of the CH2 domain; of these, only Ser239, Ser267, andHis268 were cited as part of the binding site in the Fc:FcγRIIIA crystalstructure report (Sondermann et al. EMBO J. 18: 1095-1103 (1999)). Ofthe remaining seven, a few might conceivably exert their effect byconformational change, e.g. Tyr296, Arg301, Val303, Lys338, and Asp376(FIG. 26B). However, the Glu293 and Gln295 sidechains are quite solventexposed, based on Fc crystal structures (Deisenhofer, J. Biochemistry20: 2361-2370 (1981); Guddat et al. Proc. Natl. Acad. Sci. USA90:4271-4275 (1993)), and are not involved in interactions which wouldhint at a conformational role. In addition to Glu293Ala reducing bindingby 70% (Class 8), the more conservative change of Glu293Asp also showeda similar reduction (Table 7) implying that Glu293 can indeed interactwith FcγRIIIA.

Variants which improved binding to FcγRIIIA (Classes 3, 7 and 9) includeThr256Ala, Lys290Ala, Ser298Ala, Glu333Ala, Lys334Ala and Ala339Thr. Ofthese, only Ser298 was cited as part of the binding site in theFc:FcγRIIIA crystal structure report (Sondermann et al. EMBO J. 18:1095-1103 (1999)). Though Glu333 and Lys334 do not interact withFcγRIIIA in the co-crystal structure, their interaction with FcγRIIIA issupported by four lines of evidence. First, murine IgG2b Glu333Alaexhibited a modest reduction in binding to murine FcγRII (Lund et al.Mol. Immunol. 29:53-59 (1992)). Though the same might not occur formurine FcγRIII, this shows that residues distant from the hinge regioncan influence binding to FcγR. Second, several non-Ala variants atGlu333 and Lys334 either improved or reduced binding to FcγRIIIA (Table7). Third, binding of Glu333Ala and Lys334Ala to FcγRIIIA-expressing CHOcells improved even more than seen in ELISA-based assays (Table 11).Finally, Lys334Ala exhibited a significant increase in ADCC. Thisincrease in ADCC was additive when Lys334Ala was present with Ser298Alaand was further enhanced when Glu333Ala was present (FIG. 27).

Several residues which influenced binding, albeit modestly, to FcγRIIIA,FcγRIIA and FcγRIIB, are located at the “bottom” of the CH3 domaindistant from the larger set of residues in the CH2 domain whichexhibited a more pronounced effect on binding. Lys414 (FIG. 26A) showeda 40% reduction in binding for FcγRIIA and FcγRIIB (Class 6), Arg416 a30% reduction for FcγRIIA and FcγRIIIA, Gln419 a 30% reduction toFcγRIIA and a 40% reduction to FcγRIIB, and Lys360 a 23% improvement toFcγRIIIA (Class 10). Taken together, their effect on binding of IgG1 toFcγRIIA, FcγRIIB and FcγRIIIA suggests that the “bottom” of IgG1 mayindeed be involved in the IgG1:FcγR interaction, though it may play onlya minor role.

Previous studies have mapped the binding site of murine IgG for murineFcRn (Ghetie et al. Nature Biotech. 15: 637-640 (1997); Kim et al. Eur.J. Immunol. 24: 542-548 (1994); Kim et al. Eur. J. Immunol. 24:2429-2434 (1994); Kim et al. Scand. J. Immunol. 40: 457-465 (1994);Medesan et al. J. Immunol. 158: 2211-2217 (1997)). These studies haveimplicated murine IgG residues Ile253, His310, Gln311, His433, Asn434,His435, and His436 as contacts for one FcRn molecule and Glu272 andHis285 as contacts for a second FcRn molecule. In addition, the pHdependence of the IgG:FcRn interaction has been ascribed to His310 andHis433 on IgG (as well as His250 and His251 on FcRn) (Raghavan et al.Biochemistry 34: 14649-14657 (1995)).

In the current study of the human system, a larger number of residueswere found which affected binding of IgG1 to human FcRn. Comparison ofthe human IgG1 sequence with the crystal structure of rat Fc bound tomurine FcRn (Burmeister et al. Nature 372: 379-383 (1994)) shows that inthe human Fc some of these residues could interact directly with humanFcRn: Ile253, Ser254, Lys288, Thr307, Gln311, Asn434, and His435. Nearthe Fc:FcRn interface in the crystal structure but not interactingdirectly are: Arg255, Thr256, Asp312, Glu380, Glu382, His 433, andTyr436. In the murine system it was found that altering Asn434 to Ala orGln did not affect binding to murine FcRn (Ghetie and Ward Immunol.Today 18:592-598 (1997); Medesan et al. J. Immunol. 158: 2211-2217(1997)). However, in the human system Asn434Ala exhibited the largestimprovement in binding seen for any single Ala substitution (Class 10)as well as showing additivity in combination variants (Table 8). Notethat while improvement in binding of the variants to FcRn occurred at pH7.2 as well as at pH 6.0 (Table 8), none of the variants bound well atpH 7.2. Hence, these single or combination variants may be useful inextending the half-life of human IgG1 in therapeutic antibodies, aspreviously found for murine IgG (Ghetie et al. Nature Biotech. 15:637-640 (1997)), and fulfill the requirement for binding at pH 6.0 anddissociating at pH 7.2.

Selected IgG variants were also tested for their binding to FcγRtransfected into mammalian cells. The α-chain extracellular portion ofhuman FcγRIIIA was transfected into CHO cells using a GPI-link, whereasfor human FcγRIIB the full-length receptor was transfected into CHOcells. For the variants tested, the pattern of binding to the cells wasthe same as the pattern of binding in the protein:protein (ELISA) assay(FIGS. 18A-B and 19A-B).

Combination Variants

A number of combination variants were tested in which two or moreresidues were simultaneously altered to Ala. Some of these combinationsshowed additive effects. An example is the Glu258Ala/Ser267Ala variantwhich exhibited binding to FcγRIIA and FcγRIIB that was better than theGlu258Ala (Class 4) and Ser267Ala (Class 4) variants (Tables 6 and 8). Asimilar outcome was found for the Ser298Ala/Glu333Ala andSer298Ala/Lys334Ala variants in which the binding to FcγRIIIA improvedover the parental variants (Table 8). In other combinations, one residuedominated the other, e.g. the Thr256Ala/Ser298Ala variant showed reducedbinding to FcγRIIA and FcγRIIB similar to the Ser298Ala variant eventhough the Thr256Ala change effected better binding to both thesereceptors (Class 3).

The most pronounced additivity was found for combination variants withimproved binding to FcRn. At pH 6.0, the Glu380Ala/Asn343Ala variantshowed over 8-fold better binding to FcRn, relative to native IgG1,compared to 2-fold for Glu380Ala and 3.5-fold for Asn434Ala (Tables 6and 8). Adding Thr307Ala to this effected a 13-fold improvement inbinding relative to native IgG1. Likewise, combining Glu380Ala andLeu309Ala, the latter being deleterious to FcRn binding, resulted in avariant which was intermediate between the two parental variants (Table8). As with the FcγR, some combinations showed dominance of one residueover the other; for the Lys288Ala/Asn434Ala variant, the better bindingdue to Asn434Ala clearly overcame the reduction in binding fromLys288Ala (Table 8). At pH 7.2 none of variants bound well.

Combining S298(317)A with K334(353)A improved binding to FcγRIIIA morethan either S298(317)A or K334(353)A alone (FIGS. 18A and B; and comparethe variants in Tables 6 and 8) (residue numbers in parentheses arethose of the EU index as in Kabat). Similarly, combining S298(317)A withE333(352)A improved binding to FcγRIIIA more than either S298(317)A orE333(352)A alone (compare the variants in Tables 6 and 8).

ADCC Activity of the Variants

One application of these variants is to improve the ADCC effectorfunction of an antibody. This can be achieved by modifying Fc regionamino acids at one or more residues which would lead to improved bindingto FcγRIIIA. Improved FcγRIIIA binding would lead to improved binding byNK cells, which carry only FcγRIIIA and can mediate ADCC. Selectedalanine variants which were either reduced in binding to FcγRIIIA(variants D265(278)A, E269(282)A, D270(283)A, Q295(312)A; Table 6), hadno effect on FcγRIIIA binding (R292(309)A; Table 6), or had improvedbinding to FcγRIIIA (variants K290(307)A, S298(317)A; Table 6) weretested in an in vitro ADCC assay using human PBMCs as effector cells.Since the target cells were HER2-overexpressing SKBR3 cells, the IgG Fcvariants used in this assay were generated by substituting theV_(H)/V_(L) domains of anti-IgE E27 with those from anti-HER2 antibody;HERCEPTIN® (humAb4D5-8 in Table 1 of Carter et al. PNAS (USA)89:4285-4289 (1992)). The pattern of ADCC exhibited by the variantscorrelated well with the pattern of binding to FcγRIIIA (FIGS. 20 and21). Notably the variant which showed the best improvement in binding toFcγRIIIA in protein:protein assays, variant S298(317)A, also showedimprovement in ADCC compared to wildtype HERCEPTIN® at 1.25 ng/ml (FIG.21).

One set of ADCC assays with PBMCs compared the effect of Asp265Ala(Class 1), Arg292Ala (Class 6), and Ser298Ala (Class 7). The assay wasrepeated using four different donors. FIG. 24 shows that the ADCCpattern of the variants reiterated that seen in the ELISA binding assayfor FcγRIIIA: Asp265Ala prevented ADCC (P<0.01; paired t-test),Arg292Ala had no effect, and Ser298Ala statistically improved ADCC(P<0.01; paired t-test).

A second set of assays were performed in which the FcγRIIIA allotype ofthe donors were determined. Using three FcγRIIIA-Val158/Val158 and threeFcγRIIIA-Phe158/Phe158 donors, ADCC assays using only NK cells wererepeated 3-4 times for each donor. Representative ADCC plots are shownin FIGS. 27A and 27B and the summary of all assays is shown in FIG. 27C.The variants tested were: Ser298Ala (Class 7), Lys334Ala (Class 9),Ser298Ala/Lys334Ala (Table 8), and Ser298Ala/Glu333Ala/Lys334Ala (Table8). In agreement with the binding exhibited in the ELISA-format assay(Tables 6, 8 and 11), the pattern of improved ADCC wasSer298Ala/Glu333Ala/Lys334Ala>Ser298Ala/Lys334Ala>Ser298Ala=Lys334Ala.This pattern was seen with both the FcγRIIIA-Phe158/Phe158 andFcγRIIIA-Val158/Val158 donors, though improvement in ADCC was lesspronounced for the latter. Comparing the improvement in binding toreceptor for these variants in the ELISA-format assay (Tables 6, 8 and11) with that in the cell-based (Table 11) and ADCC assays (FIGS. 24 and27) shows that the improvement in binding for any specific variant isenhanced when the receptor is expressed on cells.

EXAMPLE 5 Bind of Fc Variants to Polymorphic Fc Receptors

Allelic variants of several of the human FcγR have been found in thehuman population. These allelic variant forms have been shown to exhibitdifferences in binding of human and murine IgG and a number ofassociation studies have correlated clinical outcomes with the presenceof specific allelic forms (reviewed in Lehrnbecher et al. Blood94(12):4220-4232 (1999)). Several studies have investigated two forms ofFcγRIIA, R131 and H131, and their association with clinical outcomes(Hatta et al. Genes and Immunity 1:53-60 (1999); Yap et al. Lupus8:305-310 (1999); and Lorenz et al. European J. Immunogenetics22:397-401 (1995)). Two allelic forms of FcγRIIIA, F158 and V158, areonly now being investigated (Lehrnbecher et al., supra; and Wu et al. J.Clin. Invest. 100(5):1059-1070 (1997)). In this example, selected IgGvariants were tested against both allelic forms of FcγRIIA or FcγRIIIA.Fc receptor binding assays were performed essentially as described inthe above examples. However, for FcγRIIIA-V158, both (a) the lowaffinity receptor binding assay of Example 1 (which analyzes binding ofthe IgG complex to FcγRIIIA-V158); and (b) the high affinity FcγRbinding assay of Example 4 (which analyzes binding of IgG monomer toFcγRIIIA-V158) were carried out. The CHO stable cell lines expressingFcγRIIIA-Phe158 and FcγRIIIA-Val158 with human γ-chain were generated bysubcloning the α-chain and γ-chains into a previously described vectorwhich includes DNA encoding a green fluorescent protein (GFP) (Lucas etal. Nucl. Acids Res. 24: 1774-1779 (1996)). CHO cell transfection wascarried out using Superfect (Qiagen) according to the manufacturer'sinstructions. FACS sorting was done based on GFP expression as describedpreviously (Meng et al. Gene 242: 201-207 (2000)). Receptor expressionlevels were determined by staining with anti-FcγRIII monoclonal antibody3G8 (Medarex). Binding of IgG1 variants was performed by addingmonomeric IgG in staining buffer to 5×10⁵ cells and incubating in 96well round-bottom tissue culture plates (Costar, Cambridge, Mass.) at 4°C. for 30 min. Cells were washed three times with staining buffer andIgG binding detected by addition of 1:200 PE-F(ab′)₂ fragment of goatanti-human IgG and incubation for 30 min at 4° C. After washing,immunofluorescence staining was analyzed on a FACScan flow cytometerusing Cellquest software (Becton Dickinson). Dead cells were excludedfrom analysis by addition of 1 μg/ml propidium iodide. The results ofthese studies are summarized in Table 9-11 below.

TABLE 9 Binding of Variants to FcγRIIA and FcγRIIIA PolymorphicReceptors IgG Complex IgG Complex IgG Complex IgG Complex IgG MonomerRes#EU FCγRIIA-R131 FCγRIIA-H131 FcγRIIIA-F158 FcγRIIIA-V158FCγRIIIA-V158 IG2 (Kabat) mean sd n mean sd n mean sd n mean sd n meansd n 11 T256(269)A 1.41 (0.27) 9 1.32 (0.18) 9 0.97 (0.03) 2 1.20 1 254T256(269)N 1.03 1 1.13 1 0.95 1 0.88 1 14 D265(278)A 0.07 (0.01) 4 0.09(0.06) 4 0.01 1 15 S267(280)A 1.64 (0.18) 7 1.05 (0.03) 2 1.14 (0.25) 7189 S267(280)G 1.21 (0.05) 3 0.59 (0.09) 3 0.09 (0.02) 3 16 H268(281)A1.22 (0.14) 12 1.09 (0.01) 2 0.52 (0.09) 12 25 E283(300)A 1.24 (0.23) 51.01 (0.14) 5 0.78 1 226 E283(300)Q 1.12 1 1.19 1 0.89 1 227 E283(300)S1.03 1 0.85 1 0.83 1 228 E283(300)N 1.18 1 0.94 1 0.63 1 229 E283(300)D1.14 1 0.95 1 0.67 1 30 K290(307)A 1.29 (0.21) 7 1.28 (0.21) 7 1.12(0.05) 2 1.13 1 73 K290(307)Q 1.17 1 1.40 1 1.02 1 1.30 1 75 K290(307)S1.27 1 1.26 1 1.05 1 1.62 1 77 K290(307)E 1.10 1 1.30 1 0.98 1 1.50 1 78K290(307)R 1.05 1 1.08 1 1.07 1 1.24 1 177 K290(307)G 1.07 1 1.23 1 1.111 2.29 1 31 R292(309)A 0.27 (0.14) 9 0.90 (0.18) 9 0.94 1 80 R292(309)K0.71 (0.17) 3 1.15 (0.18) 3 1.64 1 81 R292(309)H 0.21 (0.09) 2 0.92(0.08) 2 1.21 1 82 R292(309)Q 0.47 (0.12) 3 0.45 (0.09) 3 0.56 1 83R292(309)N 0.54 (0.16) 3 0.88 (0.02) 3 0.91 1 144 E293(310)Q 0.85 (0.03)2 0.99 (0.04) 2 1.00 1 0.97 1 33 E294(311)A 0.87 (0.19) 5 0.66 (0.14) 50.68 1 173 E294(311)Q 1.01 1 0.84 1 0.79 1 174 E294(311)D 0.37 1 0.14 10.26 1 36 S298(317)A 0.40 (0.08) 12 1.30 (0.18) 12 1.02 (0.04) 2 1.96 170 S298(317)G 0.87 (0.17) 4 0.46 (0.09) 4 0.88 1 1.88 1 71 S298(317)T0.41 (0.21) 3 0.89 (0.20) 3 0.96 1 0.75 1 72 S298(317)N 0.08 (0.01) 20.06 (0.01) 2 0.66 1 0.17 1 218 S298(317)V 0.11 (0.06) 3 0.33 (0.19) 30.88 1 0.39 1 219 S298(317)L 1.14 (0.12) 3 0.34 (0.04) 3 0.83 1 0.67 140 V305(324)A 1.12 (0.12) 4 1.04 1 0.84 (0.15) 4 41 T307(326)A 1.19(0.37) 12 1.37 (0.13) 2 1.12 (0.18) 12 45 N315(334)A 1.15 (0.06) 5 1.11(0.06) 2 1.07 (0.21) 5 46 K317(336)A 1.13 (0.05) 4 1.04 1 1.10 (0.23) 448 K320(339)A 1.12 (0.11) 4 1.16 1 0.87 (0.17) 4 54 E333(352)A 0.92(0.12) 10 1.27 (0.17) 10 1.10 (0.10) 2 1.29 1 141 E333(352)Q 0.70 (0.05)2 1.10 (0.03) 2 1.05 1 1.00 1 142 E333(352)N 0.59 (0.04) 2 0.56 (0.10) 20.64 1 0.56 1 143 E333(352)S 0.94 1 0.99 1 1.07 1 152 E333(352)K 0.85(0.14) 3 0.88 1 0.81 1 153 E333(352)R 0.75 (0.04) 2 0.84 (0.05) 2 0.93 10.83 1 154 E333(352)D 1.26 (0.04) 3 1.00 1 1.70 1 178 E333(352)G 0.87 11.05 1 1.23 1 55 K334(353)A 1.01 (0.15) 17 1.39 (0.19) 17 1.07 (0.09) 31.60 (0.01) 2 135 K334(353)R 1.15 (0.09) 5 0.68 (0.07) 5 0.88 1 136K334(353)Q 1.08 (0.11) 7 1.31 (0.26) 7 1.27 (0.01) 2 1.92 1 137K334(353)N 1.16 (0.11) 7 1.15 (0.16) 7 1.19 (0.06) 2 1.70 1 138K334(353)S 1.01 (0.11) 3 1.19 (0.08) 3 1.25 1 1.82 1 139 K334(353)E 0.74(0.15) 4 1.30 (0.09) 4 1.17 1 2.75 1 140 K334(353)D 0.51 (0.09) 4 1.13(0.09) 4 1.07 1 179 K334(353)G 0.76 (0.08) 5 0.88 (0.22) 5 0.94 1 1.28 1190 K334(353)M 1.06 1 1.35 1 0.99 1 2.08 1 191 K334(353)Y 1.08 1 1.31 10.98 1 1.72 1 192 K334(353)W 0.94 1 1.07 1 0.96 1 1.53 1 193 K334(353)H1.09 1 1.26 1 0.97 1 2.06 1 220 K334(353)V 1.13 (0.11) 3 1.34 (0.18) 31.00 1 2.89 1 221 K334(353)L 1.05 1 1.38 1 0.96 1 3.59 1 65 P331(350)A1.29 (0.14) 3 1.03 (0.19) 3 0.96 1 0.78 1 198 P331(350)S 1.00 1 0.86 10.54 1 199 P331(350)N 0.86 1 0.23 1 0.24 1 200 P331(350)E 1.06 1 0.42 10.36 1 203 P331(350)K 0.94 1 0.33 1 0.26 1 96 S267(280)A 1.54 (0.12) 31.07 (0.06) 2 0.84 1 H268(281)A 110 S298(317)A 0.35 (0.13) 11 1.66(0.42) 11 1.19 (0.18) 3 E333(352)A K334(353)A 271 E380(405)A 0.98 1 0.921 1.10 1 L309(328)A

TABLE 10 Binding of Human IgG1 Variants to Human FcγRIIA-R131 andFcγRIIA-H131 Polymorphic Receptors FcγRIIA-R131^(c) FcγRIIA-R131^(d)FcγRIIIA-H131 H131/R131 Variant^(a) Class^(b) FcγR mean (sd) N mean (sd)mean (sd) mean (sd) N Ser267Ala 4 ↑II 1.52 (0.22) 11 1.53 (0.06) 1.10(0.12) 0.71 (0.07) 5 Ser267Gly ↓III 1.18 (0.10) 4 0.54 (0.14) 0.47(0.13) 5 His268Ala 5 ↑II ↓III 1.21 (0.14) 13 1.30 (0.17) 0.97 (0.15)0.75 (0.12) 10 Asp270Ala 2 ↑II, III 0.06 (0.01) 5 0.04 (0.02) 0.45(0.11) 16.6 (8.5)  6 Ser298Ala 7 ↓II ↑III 0.40 (0.15) 16 0.26 (0.10)0.24 (0.08) 0.93 (0.13) 6 Val305Ala 10 ↑FcRn 1.12 (0.12) 4 1.00 (0.14)1.06 (0.10) 1.08 (0.10) 4 Thr307Ala 4 ↑II 1.07 (0.14) 11 1.28 (0.13)1.18 (0.06) 0.94 (0.09) 5 Asn315Ala 4 ↑II 1.15 (0.06) 5 1.11 (0.18) 1.10(0.16) 0.99 (0.05) 8 Lys317Ala 10 ↑FcRn 1.13 (0.05) 4 1.10 (0.13) 1.08(0.08) 0.99 (0.07) 7 Lys320Ala no effect 1.14 (0.11) 6 1.05 (0.19) 1.13(0.09) 1.10 (0.15) 7 Ser267Ala ↑II 1.41 (0.00) 2 1.57 (0.06) 1.02 (0.08)0.65 (0.03) 4 His268Ala ^(a)Residue numbers are according to the Eunumbering system (Kabat et al. (1991) supra). ^(b)Class as noted inTable 6. ^(c)Values are from Table 6, 7 or 8. ^(d)Values are the ratioof binding of the variant to that of native IgG1 in assays separate fromthose in column 4 and performed simultaneously with the FcγRIIIA-H131assays in column 6.

TABLE 11 Binding of Human IgG1 Variants to Human FcγRIIIA-Phe158 andFcγRIIIA-Val158 Polymorphic Receptors Phe158^(c) Phe158^(d) Val158^(e)Variant^(a) Class^(b) FcγR mean (sd) N mean (sd) mean (sd) N^(f)Asp265Ala 1 ↓I, II, III 0.09 (0.06) 4 0.05 (0.02) 0.02 (0.01) 5 0.12(0.08) 0.05 (0.02) 3 Lys290Ala 3 ↑ II, III 1.31 (0.19) 9 1.15 (0.27)1.01 (0.08) 4 1.61 (0.15) 0.89 (0.04) 3 Ser298Ala 7 ↓II↑III 1.34 (0.20)16 1.49 (0.27) 1.07 (0.07) 7 1.85 (0.05) 1.18 (0.09) 3 Pro331Ala 4 ↑ II1.08 (0.19) 3 1.00 (0.23) 0.97 (0.02) 5 0.94 (0.07) 0.88 (0.09) 3Glu333Ala 9 ↓II↑III 1.27 (0.17) 10 1.13 (0.32) 1.06 (0.11) 4 1.42 (0.04)1.08 (0.09) 3 Lys334Ala 9 ↑ III 1.39 (0.19) 17 1.39 (0.22) 1.10 (0.07) 92.46 (0.08) 1.26 (0.21) 3 Ser298Ala ↓II ↑III 1.51 (0.31) 10 2.17 (0.36)1.11 (0.08) 5 Glu333Ala 3.42 (0.28) 1.65 (0.12) 3 Lys334Ala ^(a)Residuenumbers are according to the Eu numbering system (Kabat et al. (1991)supra). ^(b)Class as noted in Table 6. ^(c)Values are forFcγRIIIA-Phe158 from Table 6. ^(d)Values are the ratio of binding of thevariant to that of native IgG1 to FcγRIIIA-Phe158 in assays separatefrom those in column 4 and performed simultaneously with theFcγRIIIA-Val158 assays in column 6. Upper values are for binding in theELISA format assay; lower values are for binding to CHO cells stabletransfected with the α- and γ-chains of the receptor. ^(e)Values are theratio of binding of the variant to that of native IgG1 to FcγRIIIA-V158.Upper values are for binding in the ELISA format assay; lower values arefor binding to CHO cells stable transfected with the α- and γ-chains ofthe receptor. ^(f)Number of independent assays for values in columns 5and 6.

Selected variants were tested for binding to the FcγRIIA-H131 andFcγRIIIA-V158 allotypic receptor forms based on their improved orreduced binding to the allotypic forms used for the assays (i.e.FcγRIIA-Arg131 and FcγRIIIA-Phe158). Tables 9 and 10 show that most ofthe variants bound equivalently to the FcγRIIA-Arg131 and FcγRIIA-His131receptors. The exceptions were the Ser267Ala, His268Ala andSer267Ala/His268Ala variants which displayed binding to FcγRIIA-His131that was reduced compared to FcγRIIA-Arg131 but still equivalent tonative IgG1. The related Ser267Gly variant, however, showed a 50%reduction in binding to the FcγRIIA-His131 receptor compared to nativeIgG1. In contrast to Ser267Ala and His268Ala, Asp270Ala reduced bindingto FcγRIIA-His131 by 50% but completely abrogating binding toFcγRIIA-Arg131. This suggests that Ser267, His268 and Asp270 interactwith FcγRIIA in the vicinity of FcγRIIA residue 131.

For FcγRIIIA, the selected variants were assayed in the ELISA-format aswell as on stable-transfected CHO cell lines expressing the γ-chains(FcγRIIIA-Phe158 or FcγRIIIA-Val158) with the associated human α-chain.For FcγRIIIA-Phe158, those variants that showed improved binding in theELISA-format exhibited even more improvement, compared to native IgG1,in the cell-based assay (Table 11). This could be due to the presence ofthe γ-chain associated with the α-chain enhancing binding of the IgG toFcγRIIIA (Miller et al. J. Exp. Med. 183:2227-2233 (1996)).Alternatively, since the cell-based assay utilized monomeric IgG (incontrast to hexameric complexes used in the ELISA-format assay), thecell-based assay may be less subject to an avidity component and thusmore sensitive to changes in the binding interface. In contrast, none ofthe variants exhibited improved binding to the FcγRIIIA-Val158 receptorin the ELISA-format assay, though the Ser298Ala, Lys334Ala andSer298Ala/Glu333Ala/Lys334Ala variants did bind better than native IgG1in the cell-based assay.

Human FcγRIIA has two known, naturally occurring allotypes which aredetermined by the amino acid at position 131. Among the human IgG1variants tested against both FcγRIIA-Arg131 and FcγRIIA-His131, variantsSer267Ala, Ser267Gly, His268Ala and Asp270Ala could discriminate betweenthe polymorphic forms. This suggests that these IgG1 residues interactwith FcγRIIA in the vicinity of FcγRIIA residue 131 and in the IgG1Fc:FcγRIIIA crystal structure (Sondermann et al. Nature 406:267-273(2000)), Ser267 is adjacent to His131.

Human FcγRIIIA has naturally occurring allotypes at position 48 (Leu,His or Arg) and at position 158 (Val or Phe). The FcγRIIIA-Val158allotype binds human IgG better than the FcγRIIIA-Phe158 allotype (Koeneet al. Blood 90: 1109-1114 (1997); and Wu et al. J. Clin. Invest. 100:1059-1070 (1997)) and this difference is reiterated in the ELISA-format,cell-based and ADCC assays in this study. The IgG1 Fc:FcγRIIIA crystalstructure offers an explanation for this difference. In the crystalstructure, V158 interacts with the IgG1 lower hinge near Leu235-Gly236and with the FcγRIIIA Trp87 sidechain (which in turn interacts with theimportant IgG1 Pro329); introduction of the larger Phe158 may altereither or both of these interactions and thereby reduce the binding.

Some of the IgG1 variants exhibited better binding to FcγRIIIA-Phe158(e.g. Classes 7 and 9) and could be further improved by combiningindividual variants (Table 8). These same variants showed no or minimalimprovement in binding to FcγRIIIA-Val158 in the ELISA-format assay.However, when tested on cells expressing FcγRIIIA-Val158 or in ADCCassays using FcγRIIIA-Val158/Val158 donors, some of these variants didshow superior interaction compared to native IgG1 (Table 11, FIG. 27).Comparing the ADCC results of select IgG1 variants with better bindingto FcγRIIIA, the variants exhibited a significant improvement in ADCCfor both FcγRIIIA-Phe158/Phe158 and FcγRIIIA-Val158/Val158 donors (FIG.27). Indeed, using the Ser298Ala/Glu333Ala/Lys334Ala variant, theFcγRIIIA-Phe158/Phe158 donor ADCC could be increased over 100%(i.e. >2-fold) compared to native IgG1 (FIG. 27C).

While the influence of FcγRIIA polymorphic forms in various humandiseases has been investigated for many years (reviewed in Lehrnbecheret al. Blood 94:4220-4232 (1999)), the possible correlation betweenFcγRIIIA polymorphic forms and human disease has only recently beeninvestigated (Wu et al. J. Clin. Invest. 100: 1059-1070 (1997);Lehrnbecher et al. Blood 95: 2386-2390 (2000); Nieto et al. Arthritis &Rheumatism 43: 735-739 (2000)). Given the possible involvement of FcγRin the mechanism of action of therapeutic antibodies, human IgG1variants with improved binding capacity to human FcγR, especiallyvariants with better binding to FcγRIIIA and simultaneous abrogation ofbinding to the inhibitory FcγRIIB, could be used to provide moreefficacious therapeutic antibodies. In addition, a recent report on theoccurrence of polymorphic FcγR forms in control populations showed thatthe FcγRIIIA-Phe158 allele is more prevalent than the FcγRIIIA-Val158allele (Lehrnbecher et al. Blood 94: 4220-4232 (1999)). Since theFcγRIIIA-Phe158 receptor binds human IgG1 less well than theFcγRIIIA-Val158 receptor, therapeutic antibodies with variant Fcportions that improve binding to FcγRIIIA-Phe158 at least to the levelseen for FcγRIIIA-Val158 (if not more so) could provide increasedtherapeutic efficacy to the majority of the population.

EXAMPLE 6 Role of IgG Residues Affecting Carbohydrate

To determine whether differences in binding among variants was relatedto variation in the oligosaccharide at the conserved Asn297-linkedglycosylation site, oligosaccharides of various IgG variants wereanalyzed using matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry (MALDI-TOF-MS) as previously described(Papac et al. Glycobiology 8: 463-472 (1998)). Following immobilizationof approximately 50 μg of IgG to PVDF membranes in 96 well MultiScreen®IP plates (Millipore), proteins were reduced with 50 μL 0.1 Mdithiothreitol in 8 M urea, 360 mM Tris, 3.2 mM EDTA, pH 8.6 (RCMbuffer). Resultant free sulfhydryl groups were subsequentlycarboxymethylated by incubation with 0.1 M iodoacetic acid in RCM bufferat 25° C. for 30 min in the dark. Prior to enzymatic release ofglycoproteins, membrane-bound proteins were incubated in 1% aqueouspolyvinylpyrrolidone 360 (Sigma) solution at 25° C. for 1 hr.Oligosaccharides were released by incubating protein with 32 units ofPNGase F (New England Biolabs, Beverly, Mass.) in 25 μL of Tris-acetatebuffer, pH 8.4, at 37° C. for 3 hr, followed by acidification byaddition of 2.5 μL 1.5 M acetic acid and then incubated for 25° C. for 3hr. Samples were then purified by cation exchange chromatography usinghydrogen form, 100-200 mesh AG50W-X8 resin (Biorad). Releasedoligosaccharides were analyzed by MALDI-TOF-MS in both positive andnegative modes using matrices containing 2,5-dihydroxybenzoic acid (DHB)and 2,4,6-trihydroxyacetophenone (THAP), respectively (Papac et al.Anal. Chem. 68: 3215-3223 (1996)). Analysis was performed on a VoyagerDE mass spectrometer (PerSeptive Biosystems, Foster City, Calif.) bytransferring 0.5 μL of sample to a stainless steel target containing 0.4μL of the appropriate matrix. Following vacuum dessication, the sampleswere ionized by irradiation with an N2 laser (337 nm wavelength) andions were accelerated with a 20 kV voltage. Ion mass assignment was madeusing oligosaccharide standards (Oxford Glycosciences, Rosedale, N.Y.)in a two-point external calibration. Final spectra were the result ofthe summation of the individual spectral data from 240 laser ignitions.The results are shown in Table 12 below.

TABLE 12 Percent of Total Oligosaccharide Area by Glycan Type Glu258Asp265 Tyr296 Ser298 Arg301 Arg301 Val303 Lys334 IgG1 Ala Ala Phe AlaAla Met Ala Ala High Mannose 14.6^(a) 11.1 8.0 14.8 11.3 5.3 5.4 10.220.9 3.8 1.1 4.0 0.8 2.4 0.7 Complexes with Terminal Galactose 0 51.536.3 47.2 47.0 48.9 24.1 24.3 48.7 49.5 0.6 0.2 6.0 3.7 0.9 0.2 1 23.434.9 25.0 23.1 28.1 23.9 27.2 28.5 17.4 3.2 0.9 3.5 0.8 5.9 0.5 2 8.515.8 19.5 12.5 9.6 45.3 42.0 11.4 10.8 0.3 0.1 6.3 2.4 5.9 0.4 3 2.0 2.00.2 2.5 2.1 1.1 1.1 1.0 1.5 0.5 0.2 0.2 1.0 0.3 0.1 1-3 33.9 52.7 44.738.1 39.8 70.3 70.3 40.9 29.7 4.0 1.2 10.0 4.0 1.7 1.0 Complexes withTerminal Sialic Acid 0.0 3.0 2.4 0.0 0.0 9.7 10.9 1.3 0.0 Complexes withFucose 0 4.5 1.1 1.3 3.0 1.5 0.7 1.5 1.6 6.1 0.6 0.1 0.2 0.2 1.9 0.2 181.1 87.9 90.7 82.1 87.2 94.0 93.4 88.1 73.1 3.0 1.3 3.9 1.0 1.5 0.5Triantennary Complexes 0.2 0.2 3.2 0.0 0.0 5.5 5.0 0.3 0.0 0.2 0.2 0.11.2 0.8 0.3 ^(a)Upper values are mean percent and lower values aredeviation from mean for two independent analyses on two different lotsof IgG.

Previously it was noted that replacing human IgG3 residues which contactthe oligosaccharide, e.g. Asp265, Tyr296, Arg301, with Ala resulted inincreased galactosylation and sialylation relative to native IgG3 and inreduced binding to both FcγR and C1q (Lund et al. J. Immunol. 157:4963-4969 (1996)). In order to determine if the effect seen for specificAla substitutions (either deleterious or advantageous) was due todifferences in glycosylation, oligosaccharide analysis was performed forselected variants (Table 12). The Asp265Ala, Arg301Ala and Arg301Metvariants showed increased galactosylation, a relatively small amount ofsialylation, and a small percentage of trianntenary carbohydrate, inagreement with Lund et al., supra. The Arg301Ala and Arg301Met variantsalso showed an increase in fucose and a decrease in mannose not seenpreviously. For the Tyr296Phe variant, there were no differences fromnative IgG1, in contrast to the decrease in galactose and fucose andincrease in mannose reported by Lund et al., supra. These differencesmay be due to the different mammalian cells used to express theantibodies (human kidney 293 cells in this study and Chinese hamsterovary cells in the previous study) or may reflect that Tyr296 waschanged to Phe296 in this study whereas it was changed to Ala296 in theLund et al. study.

The Lys334 sidechain is near the carbohydrate in IgG crystal structuresbut does not interact with it as intimately as do Asp265, Tyr296 andArg301. The Lys334Ala variant exhibited a small increase in mannose andsmall decrease in fucose compared to native IgG1 (Table 12). Ser298interacts with the carbohydrate only through its Oγ atom, which forms ahydrogen bond to the Asn297 Oγ, and no difference in carbohydrate forthe Ser298Ala was evident compared to native IgG1. Neither the Glu258 orVal303 sidechains interact with the carbohydrate, indeed both arelocated on the opposite face of the CH2 domain from the carbohydrate.However, the Glu258Ala variant showed an increase in galactosylation anda small amount of sialic acid while the Val303Ala variant only showed asmall amount of sialic acid. Hence, variation in galactosylation andsialic acid for a given variant (compared to native IgG1) may occurregardless of whether the amino acid sidechain interacts with thecarbohydrate.

For the Tyr296Phe, Ser298Ala, Val303Ala and Lys334Ala variants thedifferences in glycosylation, compared to native IgG1, were minimal andmost likely were not the cause of the differences in binding of thesevariants to the FcγR. For the Glu258Ala, Asp265Ala, Arg301Ala andArg301Met variants, it is difficult to discern whether reduction orimprovement in FcγR binding is due to the change in amino acid sidechainor from differences in glycosylation.

The presence of carbohydrate linked at residue Asn297 is required forbinding to FcγR (Lund et al., supra). In addition, the nature of thecarbohydrate can influence binding (Umaña et al. Nature Biotech. 17:176-180 (1999); Lifely et al. Glycobiology 5:813-822 (1995); Lund et al.J. Immunol. 157: 4963-4969 (1996); and Lund et al. FASEB J. 9: 115-119(1995)). In crystal structures of IgG (Fc and intact antibody), Asp265interacts directly with the Asn297-linked carbohydrate via hydrogenbonds (Deisenhofer, J. Biochemistry 20: 2361-2370 (1981); Guddat et al.Proc. Natl. Acad. Sci. USA 90: 4271-4275 (1993); Harris et al. J. Mol.Biol. 275: 861-872 (1998); and Harris et al. Biochemistry 36: 1581-1597(1997)). Previous studies found that an Asp265Ala change in human IgG3altered the composition of the Asn297-linked carbohydrate and reducedbinding to FcγRI (Lund et al. J. Immunol. 157: 4963-4969 (1996); andLund et al. FASEB J. 9: 115-119 (1995)). In human IgG1, Asp265Ala(Class 1) the carbohydrate also differed from that of native IgG1 (Table12) and binding to FcγRI was reduced. Variants at position 258 and 301also showed variation from native IgG1 and the other variants (Table12). The two Arg301 variants exhibited an increase in binding toFcγRIIB, a decrease in binding to FcγRIIIA, and no effect on binding toFcγRI or FcRn (Class 5). Asp265Ala (Class 1) showed decreased binding toall FcγR while Glu258Ala (Class 4) showed increased binding to FcγRIIonly. Hence, while it is possible that the idiosyncratic carbohydrate onthese variants influenced binding rather than the amino acid changesdirectly affecting interaction with the FcγR, the data do not allowresolution of the two possibilities. For the Tyr296Phe, Ser298Ala,Val303Ala and Lys334Ala variants there were no significant differencesin carbohydrate from that of native IgG1 (Table 12). Hence thedifferences in binding to the various FcγR exhibited by these variantsis unlikely to be a result of glycosylation differences.

What is claimed is:
 1. Isolated nucleic acid encoding a variant of aparent polypeptide comprising an Fc region, wherein the Fc regionmediates antibody-dependent cell-mediated cytotoxicity (ADCC) in thepresence of human effector cells more effectively, or binds an Fc gammareceptor III (FcγRIII) with better affinity, than the parent polypeptideand wherein the Fc region comprises an amino acid modification at aminoacid position 339 and does not comprise a modification of the lowerhinge region wherein the numbering of the residues in the Fc region isthat of the EU index as in Kabat.
 2. A vector comprising the nucleicacid of claim
 1. 3. A host cell containing the vector of claim
 2. 4. Amethod for producing a polypeptide variant comprising culturing the hostcell of claim 3 so that the nucleic acid is expressed.
 5. The method ofclaim 4 further comprising recovering the polypeptide variant from thehost cell culture.
 6. The nucleic acid of claim 1 wherein the parentpolypeptide is an antibody or an immunoadhesin.
 7. The nucleic acid ofclaim 6 wherein the parent polypeptide is an antibody.
 8. The nucleicacid of claim 1 wherein the parent polypeptide Fc region comprises ahuman IgG Fc region.
 9. The nucleic acid of claim 8 wherein the humanIgG Fc region comprises a human IgG1, IgG2, IgG3 or IgG4 Fc region. 10.The nucleic acid method of claim 1 wherein the variant mediates ADCCabout 1.5 fold to about 100 fold more effectively than the parentpolypeptide.
 11. The nucleic acid of claim 1 wherein the variant bindsan FcγRIII with better affinity than the parent polypeptide.
 12. Thenucleic acid of claim 11 wherein the variant further binds an FcγRIIwith worse affinity than the parent polypeptide.
 13. The nucleic acid ofclaim 1, wherein the amino acid modification is A339T.